Membrane Damage and Protection During Freezing - Advances in

8 Membrane Damage and Protection During Freezing

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U. HEBER, H. VOLGER, V. OVERBECK, and K. A. SANTARIUS Institute of Botany, University of Dusseldorf, 4000 Dusseldorf, West Germany Freezing can damage biomembranes either mechanically or by solute action. Damage is described using mainly thylakoid membrane vesicles as examples. After freezing in suitable media, light-dependent ion gradient formation across thylakoids no longer occurs, photophosphorylation is inactivated, membrane semipermeability is lost and electron transport is either stimulated or decreased. Membranes aggregate and polar membrane proteins dissociate. A main factor in freezing damage is the action of potentially membrane-toxic solutes which are concentrated during ice formation. Membrane toxicity of salts follows a Hofmeister power series. The mechanism of membrane inactivation is discussed. Freezing damage can be prevented by adding cryoprotectants. Protection occurs by unspecific colligative solute action and by specific membrane stabilization. Sugars, sugar alcohols, and specific proteins are physiological cryoprotectants. Membranes must be protected on both sides against freeze-inactivation.

Tn

1954, L o v e l o c k r e p o r t e d t h a t h e m o l y s i s o f r e d b l o o d c e l l s , w h i c h occurred d u r i n g freezing i n physiological saline, w a s caused b y m e m -

b r a n e d a m a g e ( I ) . N o t t h e i c e crystals f o r m e d b e l o w t h e f r e e z i n g p o i n t , b u t t h e a c c u m u l a t i o n of salts a c c o m p a n y i n g i c e f o r m a t i o n w a s t h o u g h t to b e r e s p o n s i b l e f o r i n j u r y ( 2 ) .

S e v e r a l years l a t e r , p h o t o p h o s p h o r y l a -

t i o n o f t h y l a k o i d m e m b r a n e s i s o l a t e d f r o m chloroplasts of leaf cells w a s f o u n d to b e i n a c t i v a t e d d u r i n g f r e e z i n g i n t h e p r e s e n c e o f c e r t a i n solutes (3,4,5).

A g a i n , mechanical membrane damage, t h o u g h i t occurs d u r i n g 0-8412-0484-5J79J33-180-159$7.75J0 © 1979 American Chemical Society

Fennema; Proteins at Low Temperatures Advances in Chemistry; American Chemical Society: Washington, DC, 1979.

160

PROTEINS

AT

LOW

TEMPERATURES

eutectic f r e e z i n g ( 6 , 7 ) , w a s not r e s p o n s i b l e f o r loss of a c t i v i t y . chondrial oxygen uptake a n d phosphorylation, b o t h

Mito-

membrane-bound

processes, w e r e also d e c r e a s e d b y f r e e z i n g ( 8 , 9 ) . S i n c e m a n y o t h e r c e l l constituents are not a l t e r e d b y f r e e z i n g , t h e i n a c t i v a t i o n of m e m b r a n e activities suggests t h a t d a m a g e to

biomem-

branes is r e s p o n s i b l e f o r the s e n s i t i v i t y of cells a n d organisms to f r e e z i n g I n this c o n t r i b u t i o n , w e w i l l d e s c r i b e c o n d i t i o n s w h i c h l e a d to

(10-15).

m e m b r a n e d a m a g e . D a m a g e w i l l b e c h a r a c t e r i z e d u s i n g the w e l l i n v e s t i g a t e d t h y l a k o i d m e m b r a n e as a n e x a m p l e . M a n y organisms are k n o w n to a c q u i r e f r e e z i n g resistance u n d e r Downloaded by UNIV OF LIVERPOOL on March 7, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch008

s u i t a b l e e n v i r o n m e n t a l c o n d i t i o n s . O b v i o u s l y , f r e e z i n g does n o t d a m a g e their membranes.

I n v i e w of p o t e n t i a l a p p l i c a t i o n s , p a r t i c u l a r l y i n a g r i -

c u l t u r e , m e d i c i n e , a n d f o o d p r e s e r v a t i o n , i t is i m p o r t a n t to k n o w

how

m e m b r a n e d a m a g e c a n be a v o i d e d . W e w i l l b r i e f l y c o n s i d e r m e c h a n i s m s responsible for membrane protection d u r i n g freezing. Freezing I n t h e m o s t s i m p l e case, w h e n a s u s p e n s i o n of m e m b r a n e s i n a s o l u t i o n c o n t a i n i n g o n l y one solute is f r o z e n , w a t e r is c o n v e r t e d to i c e a n d the solute c o n c e n t r a t i o n rises i n the u n f r o z e n p a r t of t h e system w h i c h also contains the m e m b r a n e s . F i g u r e 1 shows t h e r e l a t i o n b e t w e e n f r e e z i n g t e m p e r a t u r e a n d t h e c o m p o s i t i o n of a b i n a r y system c o n s i s t i n g of w a t e r a n d a salt. W h e n the t e m p e r a t u r e is l o w e r e d b e l o w t h e f r e e z i n g p o i n t a n d t h e i n i t i a l salt c o n c e n t r a t i o n is n o t t o o h i g h , i c e crystals w i l l separate f r o m the s o l u t i o n . T h e m o l e f r a c t i o n of t h e salt i n the u n f r o z e n p a r t of the system increases as t h e t e m p e r a t u r e is f u r t h e r W h e n t h e eutectic t e m p e r a t u r e T m a x i m u m extent.

E

decreased.

is r e a c h e d , the system solidifies to a

A d d i t i o n of a t h i r d c o m p o n e n t to a b i n a r y m i x t u r e

shifts the eutectic t e m p e r a t u r e to l o w e r values. F u r t h e r s h i f t i n g is p r o duced

b y t h e a d d i t i o n of

more

components.

I n complex

biological

systems, w h e r e h i g h v i s c o s i t y m a y also r e t a r d c r y s t a l l i z a t i o n of solutes, eutectic f r e e z i n g is r a r e l y o b s e r v e d . W h e n i t occurs, extensive m e m b r a n e d a m a g e is p r o d u c e d , a p p a r e n t l y b e c a u s e t h e m e m b r a n e s are m e c h a n i c a l l y d i s r u p t e d b y the mass of ice a n d solute crystals f o r m e d ( 6 , 7 ) . E u t e c t i c f r e e z i n g of sea w a t e r i n w h i c h sea u r c h i n eggs w e r e s u s p e n d e d k i l l e d a l l cells

(16).

W h e n f r e e z i n g is s l o w , i c e w i l l f o r m o u t s i d e of

membrane-enclosed

spaces, a n d t h e m e m b r a n e s b e c o m e exposed to i n c r e a s e d solute c o n c e n trations.

Vesicular membranes

respond

concentrations also p r o d u c e other effects. tration of

osmotically.

Increased

solute

A s w i l l b e s h o w n , the c o n c e n -

c e r t a i n solutes is a n i m p o r t a n t f a c t o r i n t h e a l t e r a t i o n o f

b i o m e m b r a n e s d u r i n g f r e e z i n g , a n d t h i s c a n l e a d to loss of

membrane


8.

HUBER

E TA L ,

Membrane

Damage

and

161

Protection


solution

ice plus solution

\ \ \

Jsalt crystals J plus J solution

ice plus sait crystals

mole fraction of salt Figure I .

Rehtion

between temperature and composition tion. Ύ is eutectic temperature

of a single salt solu

Ε

function.

Solute d a m a g e s h o u l d , at first sight, b e e x p e c t e d t o i n c r e a s e

as t h e s u b - f r e e z i n g t e m p e r a t u r e is d e c r e a s e d because t h e solute c o n c e n t r a t i o n i n t h e u n f r o z e n p a r t of a system increases

with

decreasing

temperature. H o w e v e r , t h e changes i n membrane structure w h i c h l e a d to loss of m e m b r a n e f u n c t i o n are t e m p e r a t u r e - d e p e n d e n t processes.

Thus,

w h i l e solute stress increases as t h e t e m p e r a t u r e is l o w e r e d , t h e rate of m e m b r a n e i n a c t i v a t i o n m a y a c t u a l l y b e faster a t h i g h e r t e m p e r a t u r e s t h a n at l o w e r t e m p e r a t u r e s ( 6 ) . W h e n f r e e z i n g i s too fast t o a l l o w t r a n s p o r t of i n t r a v e s i c u l a r w a t e r to e x t r a v e s i c u l a r i c e l o c i , i c e f o r m a t i o n takes p l a c e i n s i d e m e m b r a n e e n c l o s e d spaces. T h i s appears t o b e m e c h a n i c a l l y d i s r u p t i v e . T h e r a t e of f r e e z i n g is therefore a n i m p o r t a n t p a r a m e t e r o f f r e e z i n g i n j u r y .

Slow

f r e e z i n g p r o l o n g s exposure o f m e m b r a n e s t o h i g h solute c o n c e n t r a t i o n s


162

PROTEINS

AT

LOW

TEMPERATURES

i n a t e m p e r a t u r e r a n g e w h e r e solute d a m a g e m a y p r o c e e d r a p i d l y

(6),

a n d fast f r e e z i n g m a y p r o d u c e m e c h a n i c a l m e m b r a n e d a m a g e i n c e l l u l a r systems b y i n t r a c e l l u l a r i c e f o r m a t i o n . p r o p o s e d b y M a z u r ( 17,18,19)

T h e two-stage t h e o r y of i n j u r y

describes these relations. D u r i n g f r e e z i n g

of i s o l a t e d t h y l a k o i d m e m b r a n e s s u s p e n d e d i n a n isotonic o r

somewhat

hypotonic m e d i u m , intravesicular ice formation a n d mechanical damage d o n o t o c c u r as l o n g as eutectic f r e e z i n g is a v o i d e d b y a s u i t a b l e c h o i c e of the m e d i u m a n d the final f r e e z i n g t e m p e r a t u r e . T h e f r e e z i n g d a m a g e d e s c r i b e d i n t h e f o l l o w i n g w o r k is therefore u s u a l l y c a u s e d b y h i g h solute


concentrations.

Thawing D u r i n g r a p i d t h a w i n g , membranes m a y be m e l t i n g ice.

flooded

by water from

W h e r e this occurs, osmotic e x p a n s i o n w i l l t a k e p l a c e .

If

w a t e r transport b y d i f f u s i o n is too s l o w to a b o l i s h m a j o r gradients i n t h e w a t e r p o t e n t i a l , h y p n o t i c stress is c r e a t e d i n t h e v i c i n i t y of m e l t i n g i c e , w h i l e h y p e r t o n i c stress persists i n other p a r t s of the system.

A s intact

b i o l o g i c a l m e m b r a n e s are c l o s e d a n d o s m o t i c a l l y a c t i v e , osmotic r u p t u r e c a n o c c u r w h e n h y p n o t i c o s m o t i c stress replaces solute stress. A s d u r i n g f r e e z i n g , t w o stages of i n j u r y m a y b e d i s t i n g u i s h e d . E s p e c i a l l y d u r i n g s l o w t h a w i n g , effects of h i g h solute concentrations m a y i n c r e a s e i n j u r y i n a c r i t i c a l t e m p e r a t u r e r a n g e . A l t e r n a t i v e l y , osmotic d a m a g e m a y o c c u r d u r i n g r a p i d t h a w i n g under n o n e q u i l i b r i u m conditions. S i n c e i s o l a t e d t h y l a k o i d s are less sensitive to h y p n o t i c stress t h a n i n t a c t cells, osmotic r u p t u r e does n o t o c c u r to a n y significant extent d u r i n g t h a w i n g of f r o z e n t h y l a k o i d s . Solute

Effects

During

Inorganic Salts.

Freezing R e d b l o o d cells s u s p e n d e d i n 0.15 M N a C l

first

s h r i n k d u r i n g f r e e z i n g , w h i l e the o s m o l a r i t y of the u n f r o z e n p a r t of t h e s u s p e n d i n g m e d i u m increases. W h e n i t reaches a b o u t 0.8 M, N a C l leaks i n , K is lost, a n d the cells start to h e m o l y z e o n t h a w i n g . F r e e z i n g is n o t +

necessary f o r the effect to o c c u r , as a g r a d u a l increase i n the

NaCl

c o n c e n t r a t i o n to 0.8 M or m o r e at 0 ° C also causes o s m o t i c c e l l s h r i n k a g e a n d then hemolysis ( 2 ) .

H o w e v e r , w i t h i n t a c t cells i t is r a t h e r difficult to

d e c i d e w h i c h effect p r o d u c e s i n j u r y . I n t h e c h l o r o p l a s t s of h i g h e r p l a n t s , c l o s e d c h l o r o p h y l l - c o n t a i n i n g m e m b r a n e s c a l l e d t h y l a k o i d s f u n c t i o n to c o n v e r t l i g h t e n e r g y i n t o c h e m i c a l energy. T h e y c a n b e i s o l a t e d a n d , w i t h o u t too m u c h loss of b i o c h e m i c a l a c t i v i t y , l a r g e l y f r e e d f r o m s o l u b l e c e l l constituents b y c a u t i o u s l y w a s h i n g t h e m i n h y p o t o n i c solutions o r e v e n w a t e r washing, they swell but do

n o t lose t h e i r o s m o t i c

(20,21). properties.


During When

8.

HUBER

Membrane

ET AL.

resuspended

Damage

163

and Protection

i n m e d i a c o n t a i n i n g n o n p e n e t r a t i n g solutes, t h e y

shrink.

I s o l a t e d t h y l a k o i d s c o n s t i t u t e a m u c h s i m p l e r test system t h a n i n t a c t cells for measuring the susceptibility of biomembranes to freezing. W h e n t h y l a k o i d s are f r o z e n i n a d i l u t e salt m e d i u m ( 5 m M N a C l ) , their capability to form A T P a n d A D P a n d phosphate i n t h e light is m u c h d e c r e a s e d (22,23). freezing,

I f the N a C l c o n c e n t r a t i o n is i n c r e a s e d b e f o r e

loss o f a c t i v i t y d u r i n g f r e e z i n g

is even

more

I n o r g a n i c salts s u c h as N a C l increase f r e e z i n g d a m a g e .

of f r e e z i n g , l o w concentrations o f N a C l d o n o t i n a c t i v a t e phorylation.

W h e n a cryoprotective

compound

pronounced.

I n t h e absence photophos-

is present w i t h


d u r i n g f r e e z i n g , t h e osmolar ratios o f these solutes d e t e r m i n e

NaCl

whether

m e m b r a n e f u n c t i o n is p r e s e r v e d o r lost. F i g u r e 2 shows i n a c t i v a t i o n o f t h y l a k o i d s after 3 hours of storage a t — 6 o r — 1 2 ° C as a f u n c t i o n o f the N a C l c o n c e n t r a t i o n i n the s u s p e n d i n g m e d i u m b e f o r e f r e e z i n g . C o n t r o l s w e r e k e p t f o r 3 h o u r s at 0 ° C . 100 m M

0

€ μ ι

*

= Β

20Ô

'

400

'

600

NaCl concentration Cm Ml Figure 2. Effect of temperature on inactivation of thylakoids in the presence of NaCl. Washed thylakoids were suspended in a solution containing 100 mM sucrose and NaCl and were kept for 3 hours at 0°C, — 6°C and — 12°C. Freezing and thawing were fairly rapid and final temperatures were reached within less than 2 minutes. Sucrose served as cryoprotectant and was added to prevent freeze-inactivation of the membranes in the presence of low salt concentrations. After thawing, the activity of cyclic photophosphorylation was measured. Experimental conditions have been described previously (5, 20, 21).


164

PROTEINS

A T L O W TEMPERATURES

sucrose, w h i c h is a c r y o p r o t e c t a n t i n t h e t h y l a k o i d system, w a s also a d d e d to p r e v e n t m e m b r a n e i n a c t i v a t i o n c a u s e d b y l o w concentrations o f a d d e d N a C l d u r i n g freezing.

A t —12 ° C , a n i n i t i a l c o n c e n t r a t i o n o f a b o u t 5 0

m M N a C l i n t h e s u s p e n d i n g m e d i u m w a s sufficient t o o v e r c o m e c r y o p r o t e c t i o n b y sucrose a n d p r o d u c e significant m e m b r a n e d a m a g e .

Naturally,

the N a C l concentration i n e q u i l i b r i u m w i t h ice w a s m u c h higher than the i n i t i a l c o n c e n t r a t i o n . A t — 6 ° C , a b o u t 100 m M N a C l i n t h e s u s p e n d i n g m e d i u m w e r e r e q u i r e d to p r o d u c e extensive d a m a g e .

Still higher

concentrations w e r e n e e d e d t o d a m a g e t h e m e m b r a n e s a t 0 ° C

(6,24).

T h u s , b i o m e m b r a n e s as w i d e l y d i f f e r i n g as b l o o d c e l l m e m b r a n e s a n d Downloaded by UNIV OF LIVERPOOL on March 7, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch008

p h o t o s y n t h e t i c m e m b r a n e s share a s i m i l a r s e n s i t i v i t y t o N a C l . T h e N a C l photosynthetic

membranes

share a s i m i l a r s e n s i t i v i t y t o N a C l . T h e

N a C l - i n d u c e d loss of m e m b r a n e

function during freezing or at 0 ° C

is i r r e v e r s i b l e . I n F i g u r e 2, m e m b r a n e lowered below

freezing.

damage

increases as t h e t e m p e r a t u r e is

Increased damage

is p r o d u c e d

because t h e

c o n c e n t r a t i o n o f N a C l increases p r o g r e s s i v e l y ( a b o v e t h a t s h o w n ) as t h e s u b f r e e z i n g t e m p e r a t u r e is decreased.

H o w e v e r , rates of c h e m i c a l r e a c

tions are d i r e c t l y r e l a t e d t o t e m p e r a t u r e . S o l u t e d a m a g e is n o e x c e p t i o n . It is therefore n o t s u r p r i s i n g that, as the t e m p e r a t u r e is f u r t h e r l o w e r e d below

—12 ° C , t h e extent o f solute d a m a g e m a y decrease r a t h e r t h a n

s h o w a f u r t h e r increase

(6).

1

I

1

I—'

600 ο

if α

ο

-22°C

sucrosp only o

-Vs.

- \\

-

\

·

-A0°C

\

20θί

Ο .

*

\\ η \ · \ \

1t

•·

sucrose + phenylpyruvate ^

X

-22°C 50

^

X

-30»C

v

100

150

200

hours freezing

Figure 3. Inactivation of thylakoids during freezing at various low tempera tures as a function of time. Washed thyhkoids were suspended in a solution containing 50 mM sucrose as a cryoprotectant and 20 mM sodium phenylpyru vate as a cryotoxic solute. The suspensions were rapidly frozen and thawed. After thawing, photophosphorylation was determined. For experimental condi tions, see notes in legend for Fig. 2


8.

HUBER

E T AL.

Membrane

Damage

and

165

Protection

F i g u r e 3 shows i n a c t i v a t i o n o f t h y l a k o i d s s u s p e n d e d i n a s o l u t i o n c o n t a i n i n g a c r y o t o x i c solute ( 2 0 m M s o d i u m p h e n y l p y r u v a t e ) a n d a c r y o p r o t e c t a n t ( 5 0 m M s u c r o s e ) . C o n t r o l s c o n t a i n e d sucrose o n l y . T h e r e w a s n o significant i n a c t i v a t i o n of t h y l a k o i d f u n c t i o n o f t h e controls d u r i n g 9 d a y s of storage a t — 2 2 ° C . I n t h e presence of p h e n y l p y r u v a t e , d a m a g e w a s essentially c o m p l e t e after 24 h o u r s at — 2 2 ° C . D a m a g e w a s d e c r e a s e d as t h e t e m p e r a t u r e w a s d e c r e a s e d t o — 3 0 ° C o r to — 4 0 ° C .

I n these

experiments, partial crystallization o f s o d i u m phenylpyruvate m a y have o c c u r r e d d u r i n g f r e e z i n g a n d this m a y h a v e r e d u c e d t h e effective c o n c e n t r a t i o n o f t h e c r y o t o x i c agent, p a r t i c u l a r l y a t t h e l o w e r t e m p e r a t u r e s . Downloaded by UNIV OF LIVERPOOL on March 7, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch008

T h i s effect m a y h a v e c o n t r i b u t e d t o t h e s t r i k i n g differences i n m e m b r a n e d a m a g e seen at t h e different t e m p e r a t u r e s . D u r i n g f r e e z i n g , t h y l a k o i d s suffer m o r e d a m a g e i n t h e

CATIONS.

presence of L i C l than i n t h e presence of isosmolar concentrations of N a C l ( F i g u r e 4 ) . F o r different a l k a l i m e t a l c h l o r i d e s , d a m a g e d e c r e a s e d

0

20

40

60 80 100 salt concentration C mM ]

Figure 4. The effect of different alkali metal chlorides on thylakoid function during freezing to — 20°C. Washed thylakoids were suspended before freezing in a solution containing 0.1 M sucrose and alkali metal chlorides, as indicated. The sucrose served as cryoprotectant and was added to prevent freeze-inactivation of the membranes in the presence of low salt concentrations. The suspensions were slowly frozen for 3 hours at — 20°C. After thawing in a water bath at room temperature, the activity of cyclic photophosphorylation was measured. For experimental conditions, see the legend for Fig. 2.


166

PROTEINS

i n that order: L i > +

Na

+

>

K

+

>

Rb

+

>

AT

LOW

TEMPERATURES

C s . A t 0 ° C , h i g h concentra +

tions of a l l a l k a l i m e t a l c h l o r i d e s p r o d u c e d c o m p a r a b l e m e m b r a n e i n a c t i v a t i o n (24).

S i m i l a r results h a v e b e e n o b t a i n e d d u r i n g f r e e z i n g of r e d

b l o o d cells (25). more

C h l o r i d e s of d i v a l e n t cations s u c h as C a C l

drastic membrane

(Figure 5). trations of

MgCl

2

inactivation than

chlorides of

h a d a n i n t e r m e d i a t e effect.

2

alkali

produce metals

However, l o w concen

M g , r a t h e r t h a n b e i n g t o x i c , a r e a c t u a l l y necessary + +

for


p h o t o p h o s p h o r y l a t i o n of t h y l a k o i d s .

Ο

•

ι-

20 40 60 salt concentration [mM]

Figure 5. Inactivation of thyhkoids during freezing in the presence of chlo rides of different divalent and monovalent metals. Washed thylakoids were suspended before freezing to —20°C in a solution containing 0.1 M sucrose as cryoprotectant and various chlorides at concentrations indicated on the abscissa. After thawing, the activity of cyclic photophosphorylation was measured. For experimental conditions, see legend for Fig. 2.


8.

HUBER

ET AL.

ANIONS.

Membrane

Damage

and

167

Protection

B y h o l d i n g t h e c a t i o n constant a n d v a r y i n g t h e a n i o n , t h e

effects o f different anions o n t h y l a k o i d s d u r i n g f r e e z i n g or at 0 ° C c a n b e c o m p a r e d . A s i n the case of cations, l o w c o n c e n t r a t i o n s of different anions are n o t d e s t r u c t i v e i f o c c a s i o n a l specific effects o n p h o t o p h o s p h o r y l a t i o n , s u c h as t h a t exerted b y sulfate ( 2 6 ) , are d i s r e g a r d e d ( F i g u r e 6 ) .

The

m e m b r a n e toxicities of different anions v a r y w i d e l y at h i g h concentrations. A m o n g t h e h a l o g e n i d e s , fluoride is t o l e r a t e d e v e n at r a t h e r h i g h c o n c e n trations. T o x i c i t y increases i n the o r d e r of c h l o r i d e , n i t r a t e , b r o m i d e , a n d i o d i d e [ F i g u r e 6 a n d (24)].

W i t h r e d b l o o d cells, t h e o r d e r of a n i o n

c r y o t o x i c i t y also i n c r e a s e d f r o m c h l o r i d e to i o d i d e ( 2 5 ) .

H o w e v e r , i t is


i m p o r t a n t to e m p h a s i z e t h a t m e m b r a n e i n a c t i v a t i o n is s l i g h t w h e n t h e salt c o n c e n t r a t i o n is l o w , e v e n f o r those salts t h a t are h i g h l y d e t r i m e n t a l at h i g h concentrations.

F o r e x a m p l e , c h l o r i d e at a l o w c o n c e n t r a t i o n is

a c t u a l l y necessary for t h y l a k o i d f u n c t i o n

(27).

60 80 100 salt concentration [mM] Figure 6. Cryotoxic effects of anions on thyhkoids during freezing in the presence of different sodium salts. Washed thylakoids were suspended in a solution containing 0.1 M sucrose and various sodium salts at concentrations indicated on the abscissa. For experimental conditions see legend for Fig. 2.


168

PROTEINS

O r g a n i c Salts. nontoxic

AT

LOW

TEMPERATURES

O r g a n i c salts, as is t r u e of i n o r g a n i c salts, are u s u a l l y

at v e r y l o w c o n c e n t r a t i o n s .

A t higher concentrations, their

m e m b r a n e t o x i c i t y varies g r e a t l y . F r e e z i n g r e d b l o o d cells i n the p r e s e n c e of s o d i u m acetate p r o d u c e s h e m o l y s i s (25).

Thylakoids, however,

p r o t e c t e d against f r e e z e - i n a c t i v a t i o n b y s o d i u m acetate (21).

are

T h i s effect

of s o d i u m acetate is m a r k e d l y different f r o m t h e d e t r i m e n t a l effect t h a t s o d i u m c h l o r i d e u s u a l l y has o n t h y l a k o i d s d u r i n g f r e e z i n g .

Obviously,

s o d i u m acetate is a c r y o p r o t e c t a n t f o r t h y l a k o i d s , b u t n o t f o r r e d b l o o d cells.

S o d i u m c i t r a t e , p y r u v a t e , m a l a t e , a n d tartrate are o t h e r examples

of o r g a n i c salts w h i c h c a n , a t least to some extent, p r e v e n t t h e freezeDownloaded by UNIV OF LIVERPOOL on March 7, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch008

i n a c t i v a t i o n of t h y l a k o i d s (21).

I n these instances, l o w

concentrations

are less p r o t e c t i v e t h a n h i g h c o n c e n t r a t i o n s . In

contrast to

these

salts, s o d i u m s u c c i n a t e increases t h y l a k o i d

d a m a g e d u r i n g f r e e z i n g i f p r e s e n t as t h e o n l y solute. A s w i l l b e d i s c u s s e d later, the s i t u a t i o n b e c o m e s m o r e c o m p l i c a t e d i f s i g n i f i c a n t c o n c e n t r a t i o n s of o t h e r solutes are also p r e s e n t w i t h s o d i u m s u c c i n a t e d u r i n g f r e e z i n g . M e m b r a n e i n a c t i v a t i o n b y h i g h concentrations o f s o d i u m succinate o c c u r s even atO°C

(21).

Salts of w e a k o r g a n i c acids that are s o l u b l e i n l i p i d s are also i n j u r i o u s to t h y l a k o i d s . E x a m p l e s are t h e salts of p h e n y l p y r u v i c a c i d ( F i g u r e 3 ) a n d c a p r y l i c a c i d (28).

T h e s e salts, e v e n i f present a t v e r y l o w c o n c e n

t r a t i o n s , cause extensive m e m b r a n e i n a c t i v a t i o n d u r i n g f r e e z i n g , i f c r y o protectants are absent.

A t 0 ° C , m o d e r a t e c o n c e n t r a t i o n s of these salts

w i l l slowly inactivate thylakoids. Amino Acids.

A s is t r u e of o r g a n i c a c i d s , a m i n o acids c a n e i t h e r

p r e v e n t i n a c t i v a t i o n of t h y l a k o i d s b y f r e e z i n g o r t h e y c a n a g g r a v a t e t h e s i t u a t i o n . S o m e of t h e m , f o r i n s t a n c e g l y c i n e , serine, g l u t a m a t e , or a s p a r tate, p r o m o t e i n j u r y i f p r e s e n t as the o n l y m a j o r solutes d u r i n g f r e e z i n g . H o w e v e r , the same a m i n o acids c a n b e p r o t e c t i v e i f c e r t a i n other solutes are also p r e s e n t

(28).

T h e reason f o r t h i s b e h a v i o r , w h i c h i s

also

o b s e r v e d w i t h s u c c i n a t e , w i l l be c o n s i d e r e d later. P r o l i n e , t h r e o n i n e , or γ-aminobutyric a c i d c a n p r o t e c t t h y l a k o i d s a g a i n s t i n a c t i v a t i o n d u r i n g f r e e z i n g . A m i n o acids w i t h a p o l a r side c h a i n s s u c h as p h e n y l a l a n i n e , l e u c i n e , or v a l i n e a l w a y s c o n t r i b u t e to t h y l a k o i d inactivation d u r i n g freezing. Proteins.

U s u a l l y , t h y a l k o i d s at 0 ° C c a n t o l e r a t e s o l u b l e p r o t e i n s

at h i g h concentrations b u t these p r o t e i n s a r e u s u a l l y n o t p r o t e c t i v e d u r i n g f r e e z i n g . R e m a r k a b l e exceptions are some of the p r o t e i n s f o u n d i n differ ent organs of frost-resistant p l a n t s . T h e s e p r o t e i n s c o n t a i n a h i g h p e r centage of h y d r o p h i l i c a m i n o a c i d s , are heat stable, h a v e

molecular

w e i g h t s r a n g i n g b e t w e e n 10,000 a n d 20,000 daltons, a n d are b e l i e v e d to p l a y a n i m p o r t a n t r o l e i n frost hardiness

(29,30,31).


8.

HUBER

Protection

169

A m o n g the neutral compounds,

sugars o c c u p y a

Membrane

ET AL.

N e u t r a l Solutes.

Damage

and

d o m i n a n t p o s i t i o n as f a r as p h y s i o l o g i c a l i m p o r t a n c e is c o n c e r n e d . k o i d s tolerate v e r y h i g h concentrations of sugars at 0 ° C ( 3 2 ) .

Thyla-

Freezing

i n the presence of sufficiently h i g h concentrations of h i g h l y s o l u b l e sugars s u c h as raffinose, sucrose, glucose, o r ribose does n o t l e a d to damage

(15,33).

Sugars t h u s possess c r y o p r o t e c t i v e

membrane

properties.

The

same is t r u e for sugar alcohols. H o w e v e r , p r o t e c t i o n of t h y l a k o i d s against f r e e z i n g d a m a g e c a n b e o b s e r v e d o n l y w h e n eutectic f r e e z i n g is a v o i d e d M a n n i t o l , f o r instance, is n o t effective i n p r e v e n t i n g the i n a c t i v a t i o n

(34).

of t h y l a k o i d s d u r i n g f r e e z i n g to l o w t e m p e r a t u r e s because i t c r y s t a l l i z e s Downloaded by UNIV OF LIVERPOOL on March 7, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch008

easily

(7).

G l y c e r o l is u s e d as a c r y o p r o t e c t i v e agent i n the f r e e z i n g of s p e r m a t o z o a a n d r e d b l o o d cells ( 3 5 , 3 6 , 3 7 ) . A n o t h e r c o m p o u n d t h a t has f o u n d p r a c t i c a l a p p l i c a t i o n i n t h e f r e e z e - p r e s e r v a t i o n o f cells is d i m e t h y l s u l f oxide.

B o t h g l y c e r o l a n d d i m e t h y l s u l f o x i d e c a n p r e v e n t i n a c t i v a t i o n of

thylakoids

during freezing.

Interestingly,

m e t h a n o l or e t h a n o l h a v e c r y o p r o t e c t i v e t h e y are n o t the p r e d o m i n a n t solutes During

ethylene

glycol

properties

(38),

and

even

but only if

(13).

h a r d e n i n g , m a n y p l a n t cells p r o d u c e

s u p p l y of c r y o p r o t e c t i v e c o m p o u n d s .

their o w n

internal

I n those instances, n o n p e n e t r a t i n g

s o l u b l e sugars s u c h as raffinose or sucrose are often a c c u m u l a t e d , a n d resistance to f r e e z i n g increases. Membrane Damage During Mechanical Damage

Freezing

P i e r c i n g of b i o m e m b r a n e s b y g r o w i n g i c e o r

o t h e r crystals is a s e l f - e v i d e n t means of d a m a g e .

D u r i n g fast f r e e z i n g ,

i n t r a c e l l u l a r i c e f o r m a t i o n appears to cause m e c h a n i c a l d a m a g e .

Also,

eutectic f r e e z i n g leads, e v e n i n t h e presence of a h i g h c o n c e n t r a t i o n of a c r y o p r o t e c t i v e solute, to c o m p e t e i n a c t i v a t i o n , w h i c h is p r e s u m a b l y d u e to m e c h a n i c a l d a m a g e

(7).

D u r i n g slow, natural freezing, mechanical

m e m b r a n e i n j u r y is the e x c e p t i o n , not the r u l e . Solute Injury.

A s has b e e n o u t l i n e d a b o v e , a n u m b e r of n a t u r a l

c e l l constituents are p o t e n t i a l l y h a r m f u l . I n t h e absence of f r e e z i n g a n d at p h y s i o l o g i c a l c o n c e n t r a t i o n s , t h e y are i n n o c u o u s

a n d may even

essential for the n o r m a l f u n c t i o n i n g of c e l l u l a r m e t a b o l i s m .

be

However,

d u r i n g f r e e z i n g t h e i r concentrations c a n r i s e to d a m a g i n g levels c a u s i n g i r r e v e r s i b l e alterations i n m e m b r a n e s . MEMBRANE

AGGREGATION.

I s o l a t e d t h y l a k o i d s f o r m stable s u s p e n -

sions at n e u t r a l p H , since t h e m e m b r a n e s c a r r y a net n e g a t i v e

charge.

D u r i n g t h a w i n g of t h y l a k o i d suspensions w h i c h h a v e b e e n f r o z e n i n salt solutions t h e m e m b r a n e s

aggregate

a n d precipitate.

r e d u c t i o n i n the net c h a r g e of t h e m e m b r a n e s .

T h i s indicates

a

P r e c i p i t a t i o n does n o t


170

PROTEINS

AT

LOW

TEMPERATURES

o c c u r w h e n the m e m b r a n e s are f r o z e n i n sufficiently c o n c e n t r a t e d s o l u t i o n s of sucrose o r o t h e r c r y o p r o t e c t i v e

solutes.

Usually, precipitation

indicates membrane damage. PERMEABILITY

blood

CHANGES.

AS

has b e e n m e n t i o n e d , exposure of r e d

cells sufficiently c o n c e n t r a t e d N a C l solutions at 0 ° C

f r e e z i n g w i l l r e s u l t first i n s h r i n k a g e t h e n h e m o l y s i s .

or d u r i n g

Obviously, the

c e l l u l a r m e m b r a n e b e c o m e s l e a k y a n d p e r m i t s passage

of solutes.

In

t h y l a k o i d s , A T P synthesis d u r i n g p h o t o p h o s p h o r y l a t i o n is i n a c t i v a t e d b y f r e e z i n g . I t is w i d e l y a c c e p t e d t h a t e n e r g y c o n s e r v a t i o n i n m i t o c h o n d r i a a n d chloroplasts requires membranes w i t h a l o w proton permeability. Downloaded by UNIV OF LIVERPOOL on March 7, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch008

P r o t o n transport across v e s i c u l a r m e m b r a n e s , w h i c h is c o u p l e d to e l e c t r o n f l o w ( 3 9 ) , leads to the f o r m a t i o n of t r a n s m e m b r a n e p r o t o n g r a d i e n t s , t h e e n e r g y f r o m w h i c h is b e l i e v e d to b e u s e d for the e n d e r g o n i c of A T P ( 4 0 ) .

synthesis

I n d e e d , i n t h y l a k o i d s w h i c h h a d b e e n i n a c t i v a t e d b y freez

ing, light-induced transmembrane proton

gradients

are d e c r e a s e d

a b o l i s h e d , e v e n i f e l e c t r o n t r a n s p o r t is l i t t l e affected (41,42).

or

W h e n the

m e m b r a n e s h a v e suffered o n l y m i l d d a m a g e b y f r e e z i n g , e l e c t r o n t r a n s p o r t m a y a c t u a l l y b e e n h a n c e d , w h i l e p h o t o p h o s p h o r y l a t i o n is lost U n c o u p l i n g of p h o s p h o r y l a t i o n f r o m

28).

(5,22,

e l e c t r o n t r a n s p o r t is

thus

observed. It

is n o t

Functional

only proton

p e r m e a b i l i t y t h a t changes

thylakoids respond

solutes (41,43).

osmotically

to

the

after

freezing.

a d d i t i o n of

many

T h e y s h r i n k i n h y p e r t o n i c solutions of sucrose o r N a C l

a n d e x p a n d i n h y p o t o n i c solutions.

T h e i r p e r m e a b i l i t y to cations

a n d h y d r o p h i l i c n e u t r a l m o l e c u l e s of m o d e r a t e o r l a r g e size (43) T h e y exhibit somewhat c h l o r i d e (45,46,47).

(44)

is l o w .

greater p e r m e a b i l i t y to s o m e anions s u c h as

G l y c e r o l (43)

or e t h y l e n e g l y c o l penetrate r a p i d l y .

A f t e r f r e e z i n g d a m a g e , m e m b r a n e p e r m e a b i l i t y increases i n d i s c r i m i n a t e l y . O s m o t i c responses are no l o n g e r o b s e r v e d

and the membranes

collapsed w h e n v i e w e d w i t h an electron microscope ELECTRON

TRANSPORT.

appear

(41).

I t has b e e n m e n t i o n e d t h a t m i l d

freezing

c a n i n a c t i v a t e p h o t o p h o s p h o r y l a t i o n a n d at t h e same t i m e s t i m u l a t e l i g h t dependent electron transport i n thylakoids ( 5 ) .

E l e c t r o n flow f r o m w a t e r

t h r o u g h b o t h photosystems is i n c r e a s e d i n d a m a g e d t h y l a k o i d s . H o w e v e r , after f r e e z i n g i n t h e presence of c o m p a r a t i v e l y h i g h levels of

substances

t h a t are p o t e n t i a l l y t o x i c to m e m b r a n e s , e l e c t r o n t r a n s p o r t f r o m

water

decreases a n d the w a t e r - s p l i t t i n g system b e c o m e s i n a c t i v a t e d (28).

Still,

e l e c t r o n t r a n s p o r t f r o m a d o n o r s u c h as ascorbate t h r o u g h p h o t o s y s t e m Τ

m a y b e m u c h greater i n s u c h m e m b r a n e s

thylakoids.

t h a n i t is i n f u n c t i o n a l

O n l y after v e r y severe f r e e z i n g stress is e l e c t r o n t r a n s p o r t

through photosystem Τ

decreased

(48).

T h e s e observations s h o w t h a t

the extent of m e m b r a n e d a m a g e d u r i n g f r e e z i n g d e p e n d s o n t h e f r e e z i n g c o n d i t i o n s a n d t h e solute e n v i r o n m e n t .


8.

HUBER

Membrane

ET AL.

PROTEIN RELEASE.

Damage

and

171

Protection

B i o m e m b r a n e s consist of l i p i d s a n d p r o t e i n s . T h e

latter m a y b e s u b d i v i d e d i n t o s o - c a l l e d i n t r i n s i c a n d e x t r i n s i c p r o t e i n s Intrinsic proteins supposedly

(49).

are i n t e g r a t e d i n t o t h e

phase primarily by the hydrophobic

membrane

interaction w i t h lipids.

p r o t e i n s are a t t a c h e d to t h e m e m b r a n e s .

Extrinsic

I o n i c i n t e r a c t i o n s are b e l i e v e d

to b e i m p o r t a n t i n the b i n d i n g of e x t r i n s i c p r o t e i n s . W h e n these p r o t e i n s dissociate f r o m t h e m e m b r a n e , t h e y m a y b e sufficiently h y d r o p h i l i c t o b e s o l u b l e i n the aqueous phase. W h e n freeze-aggregated

t h y l a k o i d s are

s e d i m e n t e d , a n u m b e r of m e m b r a n e proteins are f o u n d i n the s u p e r natant

fluid.

A m o n g t h e m are c a t a l y t i c p r o t e i n s i n v o l v e d i n


c o n s e r v a t i o n a n d e l e c t r o n t r a n s p o r t (42,48).

energy

T h e t o t a l a m o u n t of p r o t e i n s

r e l e a s e d d e p e n d s o n f r e e z i n g c o n d i t i o n s a n d t h e solute e n v i r o n m e n t , b u t m a y b e as m u c h as 5 %

of t h e t o t a l m e m b r a n e p r o t e i n (48).

When

f r o z e n i n the presence of a c r y o p r o t e c t i v e solute, at a sufficient c o n c e n t r a t i o n , t h y l a k o i d s r e m a i n f u n c t i o n a l a n d d o not release p r o t e i n s i n s i g n i f i cant amounts.

P r o t e i n release t h u s a c c o m p a n i e s

membrane injury and,

i n fact, is a n i n d i c a t i o n of s u c h i n j u r y . F r e e z i n g is n o t t h e o n l y cause of p r o t e i n release. P r o t e i n s dissociate from the membranes

d u r i n g i n a c t i v a t i o n of t h y l a k o i d s b y exposure

h i g h concentrations of salts at 0 ° C

(48,50).

T h e p a t t e r n of

to

membrane

p r o t e i n s released b y f r e e z i n g is s i m i l a r to the p a t t e r n of p r o t e i n s f o u n d i n the s u p e r n a t a n t fluid f r o m m e m b r a n e s w h i c h h a v e b e e n exposed h i g h salt c o n c e n t r a t i o n at 0 ° C

(Figure 7).

to a

U s u a l l y , eight bands

are

c l e a r l y a p p a r e n t i n e l e c t r o p h e r o g r a m s of p r o t e i n s released f r o m t h y l a k o i d s d u r i n g f r e e z i n g i n t h e presence of N a C l . a n d t h e y constitute m i n o r c o m p o n e n t s .

S e v e n f u r t h e r b a n d s are f a i n t T h e m o l e c u l a r w e i g h t s of

the

released p o l y p e p t i d e s r a n g e b e t w e e n 15,000 a n d 60,000 d a l t o n s . Differences i n the g e n e r a l p a t t e r n of r e l e a s e d p r o t e i n s o c c u r r e d w h e n t h y l a k o i d s w e r e f r o z e n i n solutions c o n t a i n i n g different c r y o t o x i c

com-

pounds. M o r e proteins were released w h e n thylakoids were frozen i n the presence

of

NaBr

or K B r r a t h e r t h a n N a C l .

This probably

can

be

a t t r i b u t e d to t h e greater c r y o t o x i c i t y of t h e b r o m i d e a n i o n . T h e p a t t e r n of p o l y p e p t i d e s p r o d u c e d w h e n t h y l a k o i d s w e r e f r o z e n i n the presence of sodium phenylpyruvate, sodium caprylate, or isoleucine differed greatly i n a q u a l i t a t i v e m a n n e r , a n d also to some d e g r e e i n a q u a n t i t a t i v e m a n n e r , as c o m p a r e d to t h e p a t t e r n o b t a i n e d w h e n t h e m e m b r a n e s w e r e

frozen

i n a N a C l s o l u t i o n . T h e o r g a n i c c r y o t o x i c solutes c o n t a i n i n g a p o l a r side chains released m u c h m o r e of p o l y p e p t i d e s 5 a n d 6 t h a n d i d the i n o r g a n i c salts. T h e i n t e r a c t i o n of s o d i u m p h e n y l p y r u v a t e w i t h t h e m e m b r a n e s also c a n b e d i r e c t l y o b s e r v e d at 0 ° C or 2 0 ° C b y m o n i t o r i n g s l o w changes i n l i g h t s c a t t e r i n g of the m e m b r a n e s b r o u g h t a b o u t b y the salt

(51).



172

PROTEINS

AT

LOW

TEMPERATURES

Figure 7. Electrophoretic patterns of proteins which are released from thyla koids during freezing or exposure to 0°C in the presence of various solutes. The solute concentration before freezing is indicated under the gels. Freezing time was 3 hours at — 25°C. After thawing, supernatant fluids from membranes were treated with sodium dodecylsulfate (SO) and mercaptoethanol then subjected to gel electrophoresis. Phe is sodium phenylpyruvate, Cap is sodium caprylate, lie is isoleucine. From Volger, Heber, and Berzborn (48).

I n v i e w of t h e p a r t i a l l y n o n p o l a r p r o p e r t i e s of t h e p h e n y l p y r u v a t e a n d c a p r y l a t e anions a n d of the l i p i d s o l u b i l i t y of t h e i r p r o t o n a t i o n p r o d u c t s w h i c h are i n e q u i l i b r i u m w i t h the anions, i t is l i k e l y t h a t these o r g a n i c salts not o n l y release extrinsic h y d r o p h i l i c p r o t e i n s w h i c h are a t t a c h e d to t h e m e m b r a n e s , b u t also affect h y d r o p h o b i c b o n d i n g w i t h i n t h e m e m b r a n e structure. S u c h effects c a n n o t b e seen i n p r o t e i n release experiments b e c a u s e a p o l a r proteins r e m a i n i n s o l u b l e . S o m e of t h e proteins r e l e a s e d d u r i n g f r e e z i n g h a v e b e e n i d e n t i f i e d e i t h e r b y i m m u n o e l e c t r o p h o r e t i c analysis o r b y co-electrophoresis of p u r e p r o t e i n s or p o l y p e p t i d e c h a i n s . B a n d s 1, 2, 4, 8, a n d 9 c o n t a i n t h e α, β, γ, δ a n d €-subunits of the c o u p l i n g f a c t o r C F i , r e s p e c t i v e l y .

The coupling

f a c t o r is r e s p o n s i b l e f o r t h e synthesis of A T P i n p h o t o p h o s p h o r y l a t i o n . S t i l l , t h e loss of p h o t o p h o s p h o r y l a t i o n d u r i n g f r e e z i n g i n v o l v e s m o r e t h a n just the loss of t h e c o u p l i n g f a c t o r (15,41,42,52).

Since the i o n gradients

t h o u g h t to d r i v e t h e e n d e r g o n i c synthesis of A T P c a n b e m a i n t a i n e d o n l y b y m e m b r a n e s h a v i n g a l o w i o n p e r m e a b i l i t y , t h e o b s e r v e d loss of s e m i p e r m e a b i l i t y d u r i n g f r e e z i n g is b y itself a sufficient cause f o r the i n a c t i v a t i o n of p h o t o p h o s p h o r y l a t i o n .


8.

HUBER

E T AL.

Membrane

Damage

and

173

Protection

F r o m F i g u r e 7 i t is a p p a r e n t t h a t the s u b u n i t s of C F i a p p e a r i n s o l u t i o n at v e r y different ratios d e p e n d i n g o n t h e n a t u r e of t h e c r y o t o x i c solute present d u r i n g f r e e z i n g . A n a n t i s e r u m to t h e n a t i v e c o u p l i n g factor did

not

react w i t h

the

supernatant

fluids

derived

from

membranes

d a m a g e d b y f r e e z i n g . I f C F i h a d left t h e m e m b r a n e as a n i n t a c t m o l e c u l e a n d h a d s u b s e q u e n t l y d i s s o c i a t e d i n t o s u b u n i t s as m i g h t b e

expected

f r o m its c o l d l a b i l i t y i n s o l u t i o n ( 5 3 ) , i t w o u l d h a v e g i v e n rise to b a n d patterns of

a uniform intensity distribution. T h e

observed

intensity

d i s t r i b u t i o n , w h i c h w a s v e r y different i n the presence of different c r y o toxic solutes, suggests i n s t e a d t h a t the m o l e c u l e h a d d i s i n t e g r a t e d o n the Downloaded by UNIV OF LIVERPOOL on March 7, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch008

m e m b r a n e a n d h a d released o n l y some of its s u b u n i t s . I n d e e d , the δs u b u n i t of C F w a s s h o w n b y specific antisera to b e present i n p a r t i c u l a r l y X

large amounts

i n supernatant

fluids

from

membranes

frozen

in

the

presence of s o d i u m c a p r y l a t e . W h e n t h e m e m b r a n e s w e r e f r o z e n i n t h e presence of i s o l e u c i n e , v e r y l i t t l e o f the δ-subunit, b u t a l a r g e p r o p o r t i o n of the a a n d β-subunits, w a s r e l e a s e d i n t o s o l u t i o n . I t s h o u l d b e

noted

that the k n o w n c o l d - l a b i l i t y of t h e c o u p l i n g factor is n o r m a l l y e x h i b i t e d o n l y i n s o l u t i o n , a n d not w h e n membrane.

the m o l e c u l e

is i n t e g r a t e d i n t o

the

T h e o b s e r v e d d i s i n t e g r a t i o n of the m o l e c u l e d u r i n g f r e e z i n g

therefore appears to b e p r e d o m i n a n t l y a solute effect, t h o u g h t e m p e r a t u r e m a y also p l a y a role

(15).

B a n d 2 of F i g u r e 7 w a s o c c a s i o n a l l y seen to c o n t a i n a s e c o n d

com

p o n e n t i n a d d i t i o n to the β-subunit of C F i . T h i s c o m p o n e n t is p r o b a b l y t h e l a r g e s u b u n i t of c a r b o x y d i s m u t a s e w h i c h sometimes tends to a t t a c h to t h y l a k o i d s a l t h o u g h i t is a s o l u b l e e n z y m e .

T h e s m a l l s u b u n i t of

c a r b o x y d i s m u t a s e , a n d p l a s t o c y a n i n , w h i c h w a s i d e n t i f i e d b y a specific a n t i s e r u m , are l o c a t e d i n t h e area of b a n d 9. T h e p r o t e i n of b a n d 3 is p r o b a b l y f e r r e d o x i n - N A D P - r e d u c t a s e . T h e b a n d s 5 to 7 r e m a i n u n i d e n t i fied, as are the 7 to 9 f a i n t b a n d s w h i c h are n o t v i s i b l e i n F i g u r e 7. W h i l e c o n s i d e r a b l e p r o t e i n w a s r e l e a s e d w h e n the m e m b r a n e s

were

f r o z e n a n d i n a c t i v a t e d i n the presence of salts or i s o l e u c i n e , some p r o t e i n loss is also a p p a r e n t i n the sucrose

e x p e r i m e n t of F i g u r e 7.

During

f r e e z i n g i n sucrose s o l u t i o n , the m e m b r a n e s r e m a i n e d f u n c t i o n a l . A s i n t h e salt e x p e r i m e n t s , t h e a- a n d β-subunits of t h e c o u p l i n g f a c t o r p r o m i n e n t a m o n g the c o m p o n e n t s

released i n t h e sucrose

were

experiment.

H o w e v e r , t h e r e is g o o d reason t o assume t h a t t h e p a r t i a l loss of

the

c o u p l i n g factor, w h i c h sometimes o c c u r r e d i n t h e presence of the c r y o p r o t e c t i v e agent sucrose, w a s a t t r i b u t a b l e to different causes t h a n those w h i c h l e d to d e s t r u c t i o n of the c o u p l i n g factor d u r i n g f r e e z i n g i n the presence of c r y o t o x i c salts. T h e c o u p l i n g factor c a n b e s o l u b i l i z e d a n d r e m o v e d f r o m the t h y l a k o i d s b y t r e a t m e n t w i t h E D T A , w h i c h effectively complexes

d i v a l e n t cations

(54).

E v e n w a s h i n g the membranes

salt-free sucrose solutions c a n d e t a c h t h e c o u p l i n g factor ( 5 5 ) .


with

Indeed,

174

PROTEINS

membranes

A T LOW

w a s h e d less c a r e f u l l y t h a n t h e m e m b r a n e s

TEMPERATURES

o f t h e sucrose

e x p e r i m e n t o f F i g u r e 7 s h o w e d v e r y l i t t l e loss o f p r o t e i n d u r i n g f r e e z i n g i n the p r e s e n c e o f sucrose. T h e p o s s i b i l i t y s h o u l d b e c o n s i d e r e d t h a t p r o t e i n loss d u r i n g i n a c t i v a t i o n o f t h y l a k o i d s b y f r e e z i n g results f r o m a n increase i n m e m b r a n e permeability, w h i c h w o u l d permit the leakage of intrathylakoid proteins t h r o u g h the m e m b r a n e s i n t o t h e m e d i u m . H o w e v e r , a m o n g t h e i d e n t i f i e d p r o t e i n s r e l e a s e d f r o m t h y l a k o i d s , o n l y p l a s t o c y a n i n is l o c a t e d o n t h e i n s i d e of the m e m b r a n e s .

C o u p l i n g factor C F i a n d N A D P r e d u c t a s e a r e

a t t a c h e d t o t h e o u t s i d e o f t h e t h y l a k o i d s (56,57,58,59,60). Downloaded by UNIV OF LIVERPOOL on March 7, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch008

natant

fluids

derived from membranes

W h e n super-

that h a d been inactivated b y

f r e e z i n g w e r e u s e d as i m m u n o g e n s , t h e r e s u l t i n g a n t i s e r a a g g l u t i n a t e d intact thylakoids. Since antibodies are large hydrophobic molecules w h i c h c a n n o t p e n e t r a t e b i o m e m b r a n e s , this also shows that antigens are l o c a t e d o n t h e m a t r i x side of t h y l a k o i d s (48). PROTEIN

RELEASE

IN RELATION T O

Loss

O F MEMBRANE

FUNCTION.

W h e n t h y l a k o i d s are f r o z e n i n the presence o f sucrose, m e m b r a n e f u n c t i o n is p r e s e r v e d . I f a c r y o t o x i c salt s u c h as N a C l is also present, r e t e n t i o n of m e m b r a n e f u n c t i o n a l i t y d u r i n g f r e e z i n g d e p e n d s o n t h e r a t i o o f sucrose to salt ( 5 ) . L o s s o f c y c l i c p h o t o p h o s p h o r y l a t i o n is t h e m o s t sensitive p a r a m e t e r o f m e m b r a n e i n a c t i v a t i o n . P h o t o p h o s p h o r y l a t i o n is l a r g e l y lost b e f o r e significant p r o t e i n release f r o m t h e m e m b r a n e s c a n b e d e t e c t e d ( F i g u r e 8 ) . Since photophosphorylation requires membranes w i t h u n -

l

~Ï0Ô

80 •

1

60 Ζθ • 40 mM NaCl 2

20 · 3

0 *150 4

0 mM Sucrose • 500 mM NaCl 5

Figure 8a. Fhotosystem-I-dependent phosphorylation (CPP), photosystem-Idependent electron transport (MV, methylviologen reduction in the presence of an electron donor system), and electron transport through photosystems II and I (NADP and ferricyanide reduction) in thylakoids after freezing for 3 hours to —25°C in solutions containing different ratios of sucrose to NaCl



8.

HUBER

ET AL.

Membrane

Damage

and

175

Protection

Figure 8b. Polypeptide patterns of proteins, which were released from thylakoids during freezing. The numbers 1 to 5 relate to the conditions shown along the abscissa of Fig. 8 (A). From Volger, Heber and Berzborn (48). changed

p e r m e a b i l i t y c h a r a c t e r i s t i c s , t h i s suggests

that permeability

changes o c c u r b e f o r e m u c h p r o t e i n dissociates f r o m t h e m e m b r a n e .

Thus

p r o t e i n release i n d i c a t e s a n a d v a n c e d r a t h e r t h a n a n i n i t i a l state membrane

damage.

A s photophosphorylation

decreases,

of

t h e rate

of

e l e c t r o n t r a n s p o r t increases. T h i s shows t h a t t h e loss of c o m p o n e n t s

of

t h e e l e c t r o n t r a n s p o r t c h a i n f r o m t h e m e m b r a n e s is n o t y e t c r i t i c a l , e v e n t h o u g h p r o t e i n release is a l r e a d y significant. A s the r a t i o of salt to sucrose increases, m e m b r a n e d a m a g e b e c o m e s m o r e severe a n d e l e c t r o n t r a n s p o r t is decreased.

A t the same t i m e , t h e d i s s o c i a t i o n of p r o t e i n s f r o m the

m e m b r a n e s increases c o n s i d e r a b l y . A s has b e e n m e n t i o n e d ,

components

of t h e e l e c t r o n t r a n s p o r t c h a i n s u c h as p l a s t o c y a n i n a n d N A D P r e d u c t a s e are a m o n g t h e r e l e a s e d p r o t e i n s (42,48).

I n d e e d , S t e p o n k u s et a l . ( 1 5 )

h a v e s h o w n t h a t loss of e l e c t r o n t r a n s p o r t c a n b e d e c r e a s e d b y e x p o s i n g t h y l a k o i d s to a h i g h c o n c e n t r a t i o n of p l a s t o c y a n i n d u r i n g f r e e z i n g . T h i s m i n i m i z e s loss of p l a s t o c y a n i n f r o m the m e m b r a n e s .

H o w e v e r , the con-

c l u s i o n is p r e m a t u r e t h a t p r o t e i n release is m o r e c l o s e l y r e l a t e d to i n a c t i vation

of

e l e c t r o n t r a n s p o r t t h a n to

the freeze-induced

changes

in

m e m b r a n e p e r m e a b i l i t y w h i c h cause loss of n o r m a l m e m b r a n e f u n c t i o n .


176

PROTEINS

AT

LOW

TEMPERATURES

It is r a t h e r l i k e l y that a w e a k e n i n g of i n t r a m e m b r a n e i n t e r a c t i o n s w h i c h first

causes

loss of p h o t o p h o s p h o r y l a t i o n

finally

culminates i n protein

dissociation. Mechanism

of Membrane

Damage

F a c t o r s c o n t r i b u t i n g to t h e s t a b i l i z a t i o n of b i o m e m b r a n e s are h y d r o phobic

interactions among

lipids and hydrophobic

l i p i d components

a n d between

membrane

p r o t e i n s , a n d electrostatic i n t e r a c t i o n s

among

m e m b r a n e ions a n d b e t w e e n i o n i z e d groups a n d p o l a r m o l e c u l e s .


trostatic forces a r e p a r t i c u l a r l y i m p o r t a n t i n t h e b i n d i n g of

Elec-

extrinsic

p r o t e i n s a n d i n i n t e r a c t i o n s b e t w e e n m e m b r a n e s a n d t h e solute phase. D u r i n g f r e e z i n g , w a t e r is r e m o v e d f r o m t h e m e m b r a n e suspension a n d c o n v e r t e d to ice, a n d the i o n i c s t r e n g t h increases.

Since

electrostatic

forces are not o r i e n t e d , a n increase i n the i o n i c s t r e n g t h of t h e m e d i u m w i l l finally suppress electrostatic i n t e r a c t i o n s w i t h i n t h e m e m b r a n e , p r o v i d e d ions of the solute p h a s e get close e n o u g h to the i o n i z e d m e m b r a n e sites. I n t h e i n i t i a l stages of t h e f r e e z i n g process, changes i n m e m b r a n e s t r u c t u r e are l i k e l y to o c c u r a n d this m i g h t l e a d to changes i n m e m b r a n e permeability.

A s d a m a g e progresses

a n d a sufficient n u m b e r of b o n d s

are c l e a v e d , h y d r o p h i l i c m e m b r a n e p r o t e i n s w i l l leave t h e

membrane.

T h i s process is s h o w n b e l o w f o r a s i t u a t i o n i n v o l v i n g i n t e r a c t i o n of N a C l with

noncovalent

bonds

linking

two

protein molecules

in a

single

membrane: I I

A g g r e g a t i o n of m e m b r a n e s w i l l o c c u r i f b i n d i n g of c o u n t e r i o n s

reduces

t h e net c h a r g e of m e m b r a n e s , t h e r e b y f a c i l i t a t i n g n o n c o v a l e n t i n t e r a c t i o n s b e t w e e n different m e m b r a n e s . I t is a p p r o p r i a t e t o c o n s i d e r the toxicities of different ions t o w a r d m e m b r a n e s . D u r i n g f r e e z i n g of t h y l a k o i d s , a n i o n t o x i c i t y decreases i n t h e o r d e r I " > B r " > N 0 " > C l " > F * > acetate . T h i s is r e m i n i s c e n t of t h e 3

H o f m e i s t e r l y o t r o p i c p o w e r series, w h i c h w a s o r i g i n a l l y o b s e r v e d

with

r e g a r d to d e n a t u r a t i o n of e u g l o b u l i n s , t h e n f o r b l o o d c l o t t i n g , t h e n f o r


8.

HUBER

Membrane

ET AL.

Damage

and

177

Protection

the h y d r o t h e r m a l s h r i n k a g e t e m p e r a t u r e of c o l l a g e n a n d other

effects

T h e s i m i l a r i t y b e t w e e n t h e a n i o n series t h a t f u n c t i o n s d u r i n g

(61,62, 63).

t h e f r e e z e - i n a c t i v a t i o n of

t h y l a k o i d s a n d the H o f m e i s t e r series

lends

s u p p o r t to the i d e a t h a t salt i n a c t i v a t i o n of b i o m e m b r a n e s o c c u r s t h r o u g h i n t e r f e r e n c e w i t h p o l a r b i n d i n g of m e m b r a n e c o m p o n e n t s . M a g i d (64)

Larsen and

m e a s u r e d heats of transfer of a v a r i e t y of salts f r o m w a t e r t o

solutions of m i c e l l e - f o r m i n g surfactants. T h e m i c e l l e s m a y , f o r o u r p u r pose, serve as s i m p l e m o d e l s of m e m b r a n e s .

B i n d i n g of anions to c a t i o n i c

m i c e l l e s , w h i c h i n v o l v e s c o m p e t i t i o n w i t h a n d r e p l a c e m e n t of o r i g i n a l c o u n t e r i o n s , w a s strongest for anions h a v i n g a s m a l l Stokes' l a w h y d r a t e d Downloaded by UNIV OF LIVERPOOL on March 7, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch008

r a d i u s a n d decreased w i t h i n c r e a s i n g r a d i u s . T h e o r d e r of a n i o n b i n d i n g as d e r i v e d f r o m Δ Η trans w a s B r " > citrate '.

N0 " > 3

Cl" >

F" >

acetate"

T h i s H o f m e i s t e r p o w e r series is v i r t u a l l y i d e n t i c a l w i t h

3

o r d e r of m e m b r a n e toxicities exerted b y anions i n f r e e z i n g

experiments

w i t h t h y l a k o i d s . I t is therefore c o n c l u d e d t h a t c r y o t o x i c

anions

membrane

for

binding

effects b y

sites o n

competing

w i t h membrane

the m e m b r a n e ,

thereby

anions

suppressing

> the

exert

cationic

intramembrane

interactions. M e m b r a n e i n a c t i v a t i o n d e p e n d s o n h o w closely anions c a n a p p r o a c h c a t i o n i c b i n d i n g sites. P o o r l y s o l v a t e d ions s h o w t h e strongest b i n d i n g . T h e y are also k n o w n to b e the most effective p r o t e i n dénaturants

(65).

T h e Stokes' l a w h y d r a t e d r a d i u s of the t o x i c b r o m i d e a n i o n is a b o u t A , that of t h e r e l a t i v e l y n o n t o x i c

fluoride

1.2

a b o u t 1.6 A a n d t h a t of t h e

c r y o p r o t e c t i v e acetate a n i o n 2.2 Â. B i o l o g i c a l m e m b r a n e s u s u a l l y a p p e a r i n t h i n sections as t h r e e - l a y e r e d structures 60 to 100 A t h i c k . I n v i e w of this r e l a t i v e l y l a r g e cross section, a c c e s s i b i l i t y of b i n d i n g sites b e c o m e s of obvious importance. I n t e r e s t i n g l y , the e n v e l o p e of i n t a c t chloroplasts has a n a n i o n p e r m e a b i l i t y , w h i c h f o l l o w s the order of a n i o n t o x i c i t y to t h y l a k o i d m e m b r a n e s d u r i n g f r e e z i n g . T h e i o d i d e a n i o n penetrates r a p i d l y a n d is f o l l o w e d i n order b y bromide, chloride, and penetrate

chloroplasts

slowly

fluoride

(46,66).

(Figure 9).

Acetate

S i n c e the rate of

anions

membrane

p e n e t r a t i o n b y , f o r instance, c h l o r i d e is p r o p o r t i o n a l to its c o n c e n t r a t i o n g r a d i e n t , a n d since n o c o m p e t i t i o n a p p a r e n t l y exists a m o n g v a r i o u s p e n e t r a t i n g anions, the anions diffuse across the m e m b r a n e s r a t h e r t h a n b e i n g t r a n s f e r r e d b y a c a r r i e r m e c h a n i s m . T h e a n i o n p e r m e a b i l i t y of t h y l a k o i d s seems to b e l o w e r t h a n t h a t of t h e c h l o r o p l a s t e n v e l o p e , b u t here a g a i n i o d i d e w a s f o u n d to b e t h e a n i o n that penetrates m o s t r a p i d l y

(67).

M e m b r a n e p e n e t r a t i o n b y the anions shows t h a t n o t o n l y e x t e r n a l b u t also i n t e r n a l m e m b r a n e sites are accessible f o r i n t e r a c t i o n . F o r cations, the o b s e r v e d o r d e r of t o x i c i t y d u r i n g f r e e z i n g of t h y l a k o i d s is S r

+ +

, Ca

+ +

, Ba

+ +

>

Mg

+

+

and L i

+

>

Na , K +

+

>

Rb

+

>

Cs . +

A t 0 ° C , h i g h concentrations of m o n o v a l e n t a l k a l i m e t a l cations e x h i b i t e d



178

PROTEINS

AT

LOW

TEMPERATURES

Figure 9. Change in chloroplast size on addition of various substances. Halogenides were added in 30 mM increments, which resulted in shrinkage. These additions were followed by valinomycin (val) at a concentration of 2 μΜ, which resulted in expansion. Changes in chloroplast size at 20°C were monitored by changes in the apparent absorbance of the chhroplast suspension at 535 nm (43). Note different slopes of the absorbance decrease seen on addition of valinomycin, which increases the K permeability of the chhroplast envelope. As in the presence of the .antibiotic, K diffusion is not limiting the rate of salt uptake, different slopes indicate different anion fluxes. For experimental con ditions see Kef. 91. +

+

o n l y s m a l l differences i n t h e i r m e m b r a n e toxicities (24).

T h e o r d e r of

m e m b r a n e toxicities of cations as o b s e r v e d d u r i n g f r e e z i n g of t h y l a k o i d s is s i m i l a r to the o r d e r of c a t i o n effects i n t h e l y o t r o p i c p o w e r series of H o f m e i s t e r a n d others (61,62,63).

I t is also s i m i l a r to the o r d e r i n w h i c h

cations d e n a t u r e p r o t e i n s , t h a t i s , s t r o n g l y h y d r a t e d species s u c h as L i are better dénaturants t h a n w e a k l y h y d r a t e d species s u c h as C s T h e correlation between

cationic toxicity to membranes

and

+

+

(65).

cationic

b i n d i n g to a n i o n i c surfactants is n o t as i m p r e s s i v e as i n t h e case of anions (64).

K u n t z a n d T a y l o r (65) have already noted that arguments similar

to those u s e d to e x p l a i n d i f f e r e n t i a l a n i o n b i n d i n g are n o t l i k e l y t o a p p l y


8.

HUBER

ET AL.

Membrane

Damage

and

179

Protection

to c a t i o n i c effects o n d e n a t u r a t i o n . S t i l l , i n a g r e e m e n t w i t h t h e o r d e r of i o n t o x i c i t y to m e m b r a n e s , t h e e n t h a l p y f o r t h e transfer o f salt to s o l u t i o n of s o d i u m d o d e c y l s u l f a t e w a s m o r e n e g a t i v e for C a C l M g B r , a n d that for M g B r 2

bromides

a

t h a n for

2

was more negative t h a n that for alkali m e t a l

2

(64).

I t has b e e n m e n t i o n e d t h a t t h y l a k o i d s are m o r e sensitive to salts of c e r t a i n o r g a n i c acids s u c h as p h e n y l p y r u v i c a c i d a n d c a p r y l i c a c i d t h a n to salts of i n o r g a n i c acids (28).

Differences i n the release of

proteins

a n d different p o y l p e p t i d e p a t t e r n s of r e l e a s e d p r o t e i n s ( F i g u r e 7 ) ,

(48),

suggest differences i n the a c t i o n of i n o r g a n i c a n d o r g a n i c anions. Downloaded by UNIV OF LIVERPOOL on March 7, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch008

latter b i n d to m e m b r a n e s e l e c t r o s t a t i c a l l y as w e l l as b y other

The

means.

T h i s has b e e n v e r i f i e d b y c a l o r i m e t r i c measurements of the b i n d i n g e n e r g y of the tosylate a n i o n to c a t i o n i c surfactants (64).

Depending on their

p K v a l u e s , some l o w p r o p o r t i o n of t h e p h e n y l p y r u v a t e a n d c a p r y l a t e anions are p r o t o n a t e d e v e n at n e u t r a l p H . T h e p r o t o n a t e d species

are

l i p i d s o l u b l e a n d o n f r e e z i n g t h e i r c o n c e n t r a t i o n s increase t o g e t h e r w i t h t h e concentrations of ions. T h e l i p i d - s o l u b l e m a t e r i a l w i l l a c c u m u l a t e i n t h e l i p i d p a r t of t h e m e m b r a n e p h a s e a n d d i s t u r b s h y d r o p h o b i c i n t e r actions i n the m e m b r a n e .

A s a c o n s e q u e n c e , f o r m a t i o n of

protuberances

a n d d i s i n t e g r a t i o n of m e m b r a n e s c a n be easily o b s e r v e d w i t h a m i c r o s c o p e w h e n h i g h c o n c e n t r a t i o n s of s o d i u m c a p r y l a t e a r e a d d e d t o t h y l a koids.

H y d r o p h o b i c d a m a g e to m e m b r a n e s w i l l not, h o w e v e r , b e v e r y

a p p a r e n t i n p r o t e i n release e x p e r i m e n t s , since a p o l a r p r o t e i n s a n d l i p i d s cannot b e e x p e c t e d to leave t h e m e m b r a n e p h a s e e v e n i f t h e m e m b r a n e structure is seriously d i s t u r b e d b y f r e e z i n g . P h e n y l p y r u v a t e a n d c a p r y l a t e are n o n p h y s i o l o g i c a l salts. T h u s , t h e p h y s i o l o g i c a l r e l e v a n c e of observations m a d e w i t h these c o m p o u n d s m i g h t be questioned.

H o w e v e r , p l a n t cells c o n t a i n m a n y solutes w h i c h h a v e

effects o n t h y l a k o i d s s i m i l a r to those

exerted

by

phenylpyruvate

or

c a p r y l a t e . I n c l u d e d a m o n g these c o m p o u n d s are t h e a m i n o a c i d s p h e n y l alanine,

valine, leucine,

and

isoleucine

and

a

number

of

phenolic

substances.

Membrane

Protection

Colligative Protection.

T h e p r i n c i p l e s of c o l l i g a t i v e p r o t e c t i o n w e r e

first o u t l i n e d b y L o v e l o c k ( 3 5 ) f o r t h e r e d b l o o d c e l l . T h e s e p r i n c i p l e s are also v a l i d for the t h y l a k o i d system (14,21,68). present i n a m e m b r a n e

I f o n l y one solute is

suspension, its c o n c e n t r a t i o n , regardless of its

i n i t i a l c o n c e n t r a t i o n , w i l l rise d u r i n g f r e e z i n g to a l e v e l d e t e r m i n e d solely b y the final f r e e z i n g t e m p e r a t u r e . I f t h e solute is a c r y o t o x i c

compound,

this final l e v e l m a y b e sufficient t o cause m e m b r a n e i n a c t i v a t i o n . W h e n several solutes are present a n d o n l y one is a c r y o t o x i c solute, t h e same


180

PROTEINS

AT LOW

TEMPERATURES

t o t a l o s m o l a r solute c o n c e n t r a t i o n w i l l b e o b t a i n e d w h e n s o l i d - l i q u i d e q u i l i b r i u m is a c h i e v e d at t h e same s u b f r e e z i n g t e m p e r a t u r e u s e d i n t h e first case. final

H o w e v e r , i n t h e s e c o n d case several solutes c o n t r i b u t e to the

solute c o n c e n t r a t i o n , a n d t h e c r y o t o x i c solute constitutes o n l y

f r a c t i o n of t h e t o t a l o s m o l a r c o n c e n t r a t i o n .

a

Its f r a c t i o n a l c o n c e n t r a t i o n

d e p e n d s , s i m p l y , o n the ratios of the v a r i o u s solutes e x i s t i n g i n t h e o r i g i n a l s a m p l e . I f t h e f r a c t i o n of the c r y o t o x i c solute is s m a l l , its c o n c e n t r a t i o n m a y not r e a c h a d a m a g i n g l e v e l d u r i n g f r e e z i n g . solutes t h a t " d i l u t e " t h e c r y o t o x i c c o m p o u n d

E v e n i f the n o n t o x i c

d o n o t exert a n y d i r e c t

i n f l u e n c e o n the m e m b r a n e s , t h e y w i l l nonetheless act as c r y o p r o t e c t i v e Downloaded by UNIV OF LIVERPOOL on March 7, 2017 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch008

agents. T h e s e relations m a k e i t p o s s i b l e to e x p l a i n the p a r a d o x i c a l o b s e r v a t i o n t h a t c r y o t o x i c solutes s u c h as N a C l c a n sometimes protectants d u r i n g f r e e z i n g (21).

act as

membrane

Thylakoids suspended i n a m e d i u m

c o n t a i n i n g , for i n s t a n c e , s o d i u m s u c c i n a t e as t h e p r e d o m i n a n t solute are i n a c t i v a t e d b y f r e e z i n g , b e c a u s e h i g h concentrations of s u c c i n a t e are not t o l e r a t e d b y the m e m b r a n e s ( F i g u r e 10, l e f t p a r t , I I a , b , c ) . If, h o w e v e r , i n c r e a s i n g c o n c e n t r a t i o n s of N a C l are a d d e d to t h e m e m b r a n e - s u c c i n a t e system, a r a n g e of N a C l concentrations is e n c o u n t e r e d w h e r e f r e e z i n g does n o t r e s u l t i n m e m b r a n e

damage.

A t these p a r t i c u l a r ratios

of

s u c c i n a t e to c h l o r i d e , n e i t h e r of t h e anions w i l l r e a c h d a m a g i n g levels d u r i n g f r e e z i n g a n d p r o t e c t i o n is o b s e r v e d .

T h e solute ratios, n o t t h e

i n i t i a l c o n c e n t r a t i o n s , d e t e r m i n e w h e n p r o t e c t i o n occurs.

I f the r a t i o of

N a C l to s u c c i n a t e is f u r t h e r i n c r e a s e d , f r e e z i n g w i l l raise t h e c o n c e n t r a t i o n of N a C l to a l e v e l t h a t is d a m a g i n g . S u c h observations s h o w t h a t t h e t e r m " c r y o p r o t e c t i v e a g e n t " has a v e r y loose m e a n i n g a n d does n o t necessarily i m p l y t h a t a

compound

p l a y s a n y a c t i v e r o l e i n m e m b r a n e s t a b i l i z a t i o n . W h e n a s o l u b l e sugar ( G r o u p I , F i g u r e 10)

is present i n the t h y l a k o i d suspension, f r e e z i n g

w i l l n o t cause m e m b r a n e d a m a g e , as l o n g as t h e r a t i o o f sugar to N a C l does n o t f a l l b e l o w a c r i t i c a l t h r e s h o l d v a l u e , b e c a u s e e v e n h i g h c o n c e n trations of sugars are t o l e r a t e d b y t h e m e m b r a n e s . Specific Protection.

Low

MOLECULAR

WEIGHT

SOLUTES.

Colliga-

t i v e p r o t e c t i o n is nonspecific. A n y solute w h i c h does n o t d a m a g e a b i o m e m b r a n e m u s t , just b y its presence,

r e d u c e t h e c o n c e n t r a t i o n of

a

p o t e n t i a l l y c r y o t o x i c solute d u r i n g f r e e z i n g as c o m p a r e d to t h e c o n c e n t r a t i o n the c r y o t o x i c solute w o u l d a t t a i n i n the absence of a s e c o n d solute. O n a n o s m o l a r basis, different n e u t r a l solutes s h o u l d b e e q u a l l y effective. H o w e v e r , t h e facts differ f r o m t h i s e x p e c t a t i o n .

O n an

basis, raffinose is a b e t t e r c r y o p r o t e c t a n t t h a n sucrose

equi-osmolar

is s u p e r i o r to

glucose, a n d g l u c o s e is m o r e effective t h a n g l y c e r o l (15,33,34).

Similar

d e v i a t i o n s f r o m " i d e a l " c o l l i g a t i v e b e h a v i o r h a v e b e e n o b s e r v e d f o r other solutes (68).

T o e x p l a i n the differences, i t is necessary to e i t h e r i n t r o d u c e



8.

HUBER

Membrane

E T AL.

Damage

and

181

Protection

N a C l concentration Figure 10. Thylakoid protection by two different groups of compounds as a function of the concentration of a potentially cryotoxic solute such as NaGL Examples of solutes belonging to group! are soluble sugars, sugar alcohols, proline, threonine. Representatives of group II include sodium succinate, glutamate and asparate. a, b, c, are different concentrations increasing from a to c. The osmolar ratio of a given compound from group I or II versus NaCl, that is needed to produce 50% inactivation of the membrane during freezing, differs depending on the compound (not indicated in this figure). o s m o t i c coefficients o r t o assume t h a t solutes h a v e d i r e c t solute-specific effects o n m e m b r a n e s

i n a d d i t i o n t o t h e i r c o l l i g a t i v e effects.

s t a b i l i z a t i o n c a n i n d e e d b e o b s e r v e d i n t h e absence

Direct

of f r e e z i n g , w h e n

c o l l i g a t i v e d i l u t i o n of m e m b r a n e - t o x i c solutes is n o t p o s s i b l e . F o r e x a m p l e , t h y l a k o i d s s u s p e n d e d i n N a C l solutions a t 0 ° C are i n a c t i v a t e d faster i n t h e absence dimethyl

o f sucrose

sulfoxide

t h a n i n its p r e s e n c e

c a n protect

unfrozen

(21).

Furthermore,

o v a r y cells o f t h e C h i n e s e

h a m s t e r against d a m a g e b y a h y p e r o s m o t i c salt s o l u t i o n (69).

Molecular

details o f specific m e m b r a n e p r o t e c t i o n are n o t y e t k n o w n . I t is p o s s i b l e t h a t effects o n w a t e r s t r u c t u r e are i n v o l v e d . CRYOPROTECTTVE

PROTEINS.

I n general, soluble proteins are either

w e a k l y effective or ineffective f o r p r e v e n t i n g t h e f r e e z e - i n a c t i v a t i o n of t h y l a k o i d s s u s p e n d e d i n d i l u t e salt solutions. T h i s is n o t u n e x p e c t e d since l o w concentrations of h i g h m o l e c u l a r w e i g h t c o m p o u n d s s u c h as p r o t e i n s c a n n o t s i g n i f i c a n t l y r e d u c e t h e f r e e z e - c o n c e n t r a t i o n of c r y o t o x i c solutes by

colligative action.

H o w e v e r , some p r o t e i n s

extracted f r o m


frost-

182

PROTEINS

AT

LOW

TEMPERATURES

resistant p l a n t s c a n , e v e n at v e r y l o w c o n c e n t r a t i o n s , exert a p r o t e c t i v e effect o n t h y l a k o i d s d u r i n g f r e e z i n g . A t c o n c e n t r a t i o n s of less t h a n 40 μΜ, these p r o t e i n s p r o d u c e as m u c h m e m b r a n e p r o t e c t i o n as sucrose at a c o n c e n t r a t i o n of 3 0 m M ( c a l c u l a t e d f r o m refs. 31 a n d 7 0 ) .

T h e s e facts

a l o n e m a k e p r o t e c t i o n o n a c o l l i g a t i v e basis h i g h l y u n l i k e l y f o r p r o t e i n s . T h e p r o t e i n s are h e a t stable a n d w a t e r s o l u b l e .

T h e amino

com

p o s i t i o n s o f t w o of t h e m h a v e b e e n d e t e r m i n e d , a n d t h e y c o n t a i n h i g h percentages of p o l a r a m i n o a c i d s a n d l o w percentages of a m i n o a c i d s w i t h n o n p o l a r side c h a i n s .


L i t t l e is k n o w n c o n c e r n i n g m e c h a n i s m s b y w h i c h these p r e v e n t i n a c t i v a t i o n of t h y l a k o i d s d u r i n g f r e e z i n g , b u t t h e y

proteins somehow

c o n t r i b u t e to m e m b r a n e s t a b i l i z a t i o n . T h e y act w i t h some specificity, since c r y o p r o t e c t i v e p r o t e i n s f r o m s p i n a c h not o n l y f a i l t o p r o t e c t r e d b l o o d cells d u r i n g f r e e z i n g b u t are a c t u a l l y i n j u r i o u s . A b i l i t y of Cryoprotectants to Penetrate Membranes.

Preserving

n o r m a l m e m b r a n e p r o p e r t i e s d u r i n g f r e e z i n g poses s p e c i a l p r o b l e m s . L o v e l o c k (71)

has r e p o r t e d t h a t o n l y n e u t r a l h y d r o p h i l i c m o l e c u l e s of a

size s m a l l e n o u g h to p e n e t r a t e m e m b r a n e s

were

successful

cryopro

tectants f o r r e d b l o o d cells s u s p e n d e d i n p h y s i o l o g i c a l saline. T h i s s u g gests t h a t not o n l y t h e o u t e r b u t also t h e i n n e r side of t h e c e l l u l a r membrane requires protection.

M o r e recent w o r k ( 9 2 )

has s h o w n t h a t

the i n n e r s i d e is less sensitive to f r e e z i n g d a m a g e t h a n the o u t e r p e r h a p s because solutes o f

t h e i n n e r p h a s e c o n t r i b u t e to

one,

protection.

P r o v i d e d the i n t e r i o r of a c e l l contains a n excess o f a c r y o t o x i c solute, p r o t e c t i o n is p o s s i b l e o n l y i f i t c a n l e a k o u t d u r i n g f r e e z i n g t o

be

colligatively d i l u t e d b y a nonpenetrating cryoprotectant located outside, o r i f a p e n e t r a t i n g c r y o p r o t e c t a n t c a n enter t h e c e l l .

If b o t h

cannot

p e n e t r a t e , t h e y m u s t b e p r e s e n t o n t h e same m e m b r a n e side f o r p r o t e c t i o n to b e c o m e p o s s i b l e . I n p l a n t cells, a c c u m u l a t i o n of s o l u b l e sugars has often b e e n n o t i c e d d u r i n g h a r d e n i n g a n d this has b e e n suggested as a f a c t o r i n frost h a r d i ness (12,14,68).

E x t r a c e l l u l a r a d d i t i o n of sugars to frost-sensitive cells

fails t o p r o t e c t against f r e e z i n g i n j u r y (72,73) s u g a r u p t a k e w a s s u b s t a n t i a l (74,75).

e x c e p t i n cases w h e r e

I n v i e w of this, i t is s u r p r i s i n g

t h a t t h y l a k o i d s s u s p e n d e d i n solutions c o n t a i n i n g c r y o t o x i c solutes c a n b e p r o t e c t e d against f r e e z e - i n a c t i v a t i o n n o t o n l y b y p e n e t r a t i n g solutes s u c h as g l y c e r o l or d i m e t h y l s u l f o x i d e , b u t also b y r e l a t i v e l y l a r g e sugars such

as

glucose,

sucrose,

or

raffinose

r e g a r d e d as n o n p e n e t r a t i n g (43).

(15,33),

w h i c h are n o r m a l l y

A s mentioned above, even some p r o

teins w i t h m o l e c u l a r w e i g h t s of 10,000-20,000 d a l t o n s p r o t e c t t h y l a k o i d s against f r e e z i n g d a m a g e , a l t h o u g h c o m p l e t e p r o t e c t i o n i s n o t unless other c r y o p r o t e c t a n t s are p r e s e n t (

31).


observed

8.

HUBER

Membrane

ET AL.

Damage

and

183

Protection

P r o t e c t i o n of t h y l a k o i d s b y l a r g e m o l e c u l e s m i g h t b e e x p l a i n e d i n several ways.

P e r h a p s the t h y l a k o i d m e m b r a n e

is sensitive to

solute

i n j u r y o n l y o n the o u t e r side, or the i n t r a t h y l a k o i d space contains, e v e n after l o n g i n c u b a t i o n , l i t t l e of t h e a d d e d c r y o t o x i c solute, or p e n e t r a t i o n of t h e m e m b r a n e b y n o r m a l l y n o n p e n e t r a t i n g solutes b e c o m e s p o s s i b l e under freezing conditions

(76).

T h e fact t h a t c r y o p r o t e c t i v e proteins alone cannot p r o v i d e

complete

p r o t e c t i o n to t h y l a k o i d s d u r i n g f r e e z i n g , e v e n w h e n present at s a t u r a t i n g concentrations, c a n b e r e g a r d e d as e v i d e n c e t h a t n o t o n l y t h e o u t s i d e b u t also t h e i n s i d e of t h e t h y l a k o i d m e m b r a n e r e q u i r e s p r o t e c t i o n .

When


f r o z e n i n a sucrose s o l u t i o n of sufficient c o n c e n t r a t i o n , t h y l a k o i d s are completely protected.

A l t h o u g h sugars, w h e n first a d d e d to t h y l a k o i d

m e m b r a n e s , p r o d u c e a n osmotic response as e x p e c t e d f r o m v a n t H o f F s l a w , t h e y a p p a r e n t l y l e a k i n t o t h e i n t r a t h y l a k o i d space d u r i n g f r e e z i n g ( 7 6 ) , t h e r e b y p r o v i d i n g p r o t e c t i o n to the i n n e r side of the m e m b r a n e . I t is n o t k n o w n w h e t h e r c r y o t o x i c solutes l e a k c o n c u r r e n t l y o u t f r o m t h e i n t r a t h y l a k o i d space.

O b v i o u s l y , the p e r m e a b i l i t y p r o p e r t i e s of b i o m e m -

branes h a v e a n i m p o r t a n t r o l e i n m e m b r a n e p r o t e c t i o n . S i n c e the p e r m e a b i l i t y p r o p e r t i e s of a m e m b r a n e d e p e n d o n m e m b r a n e s t r u c t u r e , a n y s t r u c t u r a l changes s h o u l d influence m e m b r a n e s u r v i v a l d u r i n g f r e e z i n g . Protection by Changes i n M e m b r a n e S t r u c t u r e .

Development

of

frost h a r d i n e s s i n p l a n t s is o f t e n a c c o m p a n i e d b y a n i n c r e a s e i n m e m b r a n e l i p i d s , p a r t i c u l a r l y p h o s p h o l i p i d s (77, 78, 7 9 ) .

A n increase i n the d e g r e e

of u n s a t u r a t i o n of t h e f a t t y a c i d c o m p o n e n t s

of p h o s p h o l i p i d s also has

been

observed

by

some

(78,79)

b u t not b y

A t t e m p t s to correlate increases i n m e m b r a n e

a l l investigators

t h y l a k o i d s f r o m c a b b a g e leaves h a v e so f a r f a i l e d ( 8 0 ) . an increase

i n membrane

lipids may

have

(77).

l i p i d s w i t h hardiness on

of

A l s o , w h a t effect

h a r d i n e s s is n o t

yet

understood. I t is k n o w n t h a t h a r d y p l a n t cells e x h i b i t i n c r e a s e d p e r m e a b i l i t y to water

(12,81,82,83).

differences

I n some cases,

i n chloroplasts

r e p o r t e d (15,84,85).

cytological

and

of h a r d y a n d n o n h a r d y

ultrastructural

leaves

have

been

T h e s e observations r e q u i r e i n t e r p r e t a t i o n .

B y means of s u i t a b l e m e m b r a n e - a c t i v e a d d i t i v e s , w e h a v e a t t e m p t e d to m o d i f y the s t r u c t u r e of i s o l a t e d t h y l a k o i d s a n d t h e r e b y i n c r e a s e m e m b r a n e p e r m e a b i l i t y . C h a n g e s i n resistance of t h e m e m b r a n e s to f r e e z i n g w e r e t h e n d e t e r m i n e d . F i g u r e 11 shows t h e effect of s o d i u m c a p r y l a t e o n t h e p e r m e a b i l i t y of t h y l a k o i d s to protons. p o u n d , i l l u m i n a t i o n causes

I n t h e absence of t h e

com-

p r o t o n transfer across the t h y l a k o i d s a n d

a c i d i f i c a t i o n of t h e i n t r a t h y l a k o i d space.

A

fluorescent

u s e d to m o n i t o r f o r m a t i o n of t h e p r o t o n g r a d i e n t ( 8 6 ) .

weak amine was W h e n the light

w a s t u r n e d off, p r o t o n s m o v e d s l o w l y b a c k across t h e t h y l a k o i d m e m b r a n e . T r a n s p o r t f o l l o w e d first-order k i n e t i c s . I n t h e presence of c a p r y l a t e


184

PROTEINS A T

LOW

TEMPERATURES

5 AJM 9-aminoacridine

ι


ο c

,

mM Na-caprylate

,

20

1 min

Figure 11. Formation of a trans-thyhkoid proton gradient by intact chloro plasts as indicated by the quenching of 9-aminoacridine fluorescence (86, 87), and a subsequent efflux of protons from the thylakoids on darkening. Note accelerated proton efflux in the presence of sodium caprylate. Conditions: Intact spinach chloroplasts were suspended in isotonic sorbitol buffer (20 μg chloro phyll ml' ) and illuminated with saturating red light in the presence of 0.5 mM methyhiologen as electron acceptor. 1

o r other s e m i p o l a r c o m p o u n d s

of s u i t a b l e s t r u c t u r e a n d c o n c e n t r a t i o n ,

p r o t o n p u m p i n g s t i l l p r o d u c e d l a r g e p r o t o n g r a d i e n t s i n the l i g h t .

How

ever, w h e n t h e l i g h t w a s t u r n e d off, p r o t o n s l e a k e d o u t c o n s i d e r a b l y faster t h a n t h e y d i d i n t h e absence

of p h e n y l p y r u v a t e . T h e a v a i l a b l e

d a t a suggest t h a t salts h a v i n g a n a n i o n t h a t c a n b i n d to c a t i o n i c m e m b r a n e sites a n d exert h y d r o p h o b i c effects w i l l increase the p r o t o n c o n d u c t i v i t y of t h y l a k o i d s . L i g h t s c a t t e r i n g m e a s u r e m e n t s i n d i c a t e t h a t the p e r m e a b i l i t y to sugars a n d other c o m p o u n d s is also i n c r e a s e d

(51).

F i g u r e 12 shows p r e s e r v a t i o n of m e m b r a n e f u n c t i o n d u r i n g f r e e z i n g of

thylakoids i n the presence

of

different c o n c e n t r a t i o n s

of

sodium

c a p r y l a t e o r s o d i u m p h e n y l p y r u v a t e . O t h e r solutes p r e s e n t i n t h e system


8.

HUBER

E T AL.

Membrane

Damage

and


τ

185

Protection ρ

Figure 12. Preservation of thylakoid function after freezing for 3 hours at —25°C in the presence of different concentrations of sodium phenylpyruvate (O) or sodium caprylate (Φ). Other solutes added to the thylakoid suspension before freezing were, 100 mM NaCl and 120 mM sucrose in the caprylate ex periment and 100 mM NaCl and 150 mM sorbitol in the phenylpyruvate experi ment. were sorbitol and N a C l .

T h e i r m o l a r r a t i o w a s k e p t constant at a l e v e l

p r o d u c i n g c o n s i d e r a b l e i n a c t i v a t i o n d u r i n g f r e e z i n g to

— 25 ° C i n the

absence of p h e n y l p y r u v a t e or c a p r y l a t e . W h e n p h e n y l p y r u v a t e o r c a p r y l a t e w a s a d d e d at a v e r y l o w c o n c e n t r a t i o n , a significant increase w a s o b s e r v e d i n the resistance of t h e m e m b r a n e s t o f r e e z i n g . P r o t e c t i o n w a s o p t i m a l i n the presence

of 2 m M c a p r y l a t e o r 4 m M p h e n y l p y r u v a t e .

F u r t h e r increases i n c o n c e n t r a t i o n first d e c r e a s e d p r o t e c t i o n a n d

finally

l e d to c o m p l e t e m e m b r a n e i n a c t i v a t i o n d u r i n g f r e e z i n g . W h e n t h e i n i t i a l c o n c e n t r a t i o n of s o r b i t o l a n d N a C l w a s h i g h e r t h a n t h a t u s e d f o r t h e experiment

of

F i g u r e 12, a c o r r e s p o n d i n g l y

higher concentration

of

s o d i u m p h e n y l p y r u v a t e or c a p r y l a t e w a s necessary to a c h i e v e m a x i m u m protection d u r i n g freezing.

Thus, depending on concentration, p h e n y l -

p y r u v a t e , a n d c a p r y l a t e a c t e d either as cryoprotectants o r as solutes.

cryotoxic

S u i t a b l e controls e s t a b l i s h e d t h a t c r y o p r o t e c t i o n b y these

p o u n d s w a s not b a s e d o n c o l l i g a t i v e a c t i o n .

com

F o r instance, the sorbitol

c o n c e n t r a t i o n h a d t o b e r a i s e d b y a b o u t 50 m M t o get t h e same p r o t e c t i o n that was produced b y 3 m M phenylpyruvate. F u r t h e r m o r e , p r o t e c t i o n is n o t h i g h l y specific, since i s o l e u c i n e a n d s o d i u m d e c e n y l s u c c i n a t e p r o t e c t e d t h e m e m b r a n e s to essentially t h e same extent as c a p r y l a t e o r p h e n y l p y r u v a t e . H o w e v e r , m e m b r a n e p r o t e c t i o n i n the presence of p h e n y l p y r u v a t e is d e p e n d e n t o n the c o m p o s i t i o n of t h e


186

PROTEINS

m e d i u m i n w h i c h the thylakoids are suspended.

A TL O W TEMPERATURES

Protection has been

o b s e r v e d i n solutions c o n t a i n i n g b a l a n c e d concentrations o f N a C l a n d e i t h e r s o r b i t o l ,s u c r o s e , t h r e o n i n e ,o r s o d i u m s u c c i n a t e (51, 68). T h e p e r m e a b i l i t y o f t h y l a k o i d s t o these c o m p o u n d s

is low. Phenylpyruvate

fails t o increase m e m b r a n e p r o t e c t i o n i n solutions c o n t a i n i n g N a C l a n d glycerol or methanol. T h e latter compounds penetrate thylakoids r a p i d l y . T h e s e d a t a suggest t h a t t h e i n c r e a s e d p r o t e c t i o n seen i n the p r e s e n c e o f l o w concentrations o f s e m i p o l a r s o l u t e s ,s u c h as s o d i u m p h e n y l p y r u v a t e , of n o r m a l l y n o n p e n e t r a t i n g c r y o p r o t e c t a n t s t o t h e i n s i d e o f t h e m e m b r a n e s


w h e r e p r o t e c t i o n is also r e q u i r e d . N a t u r a l l y ,t h e m a n i p u l a t i o n o f m e m b r a n e p e r m e a b i l i t y is a dangerous m a t t e r ,since m e m b r a n e f u n c t i o n is i n t i m a t e l y r e l a t e d t o m e m b r a n e p e r m e ability.

M a n i p u l a t i o n t h a t goes t o o f a r c a n e a s i l y cause

membrane

d a m a g e , as occurs i n t h e p r e s e n c e o f i n c r e a s e d c o n c e n t r a t i o n s o f c a p r y l a t e a n d p h e n y l p y r u v a t e ( F i g u r e 1 2 ) . H i g h c o n c e n t r a t i o n s o f these p o u n d s also cause r a p i d m e m b r a n e i n a c t i v a t i o n a t 0 ° C .

com

S t i l l ,manipula

t i o n o f m e m b r a n e p e r m e a b i l i t y as a means o f i n c r e a s i n g resistance t o f r e e z i n g appears p o s s i b l e i n p r a c t i c e . F o r e x a m p l e ,K u i p e r (88) r e p o r t e d t h a t s o d i u m d e c e n y l s u c c i n a t e i n c r e a s e d frost h a r d i n e s s i n p l a n t s . T h a t other w o r k e r s h a v e f a i l e d t o observe p r o t e c t i o n against f r e e z i n g d a m a g e b y d e c e n y l s u c c i n a t e a n d i n s t e a d h a v e r e p o r t e d i n c r e a s e d d a m a g e (89, 90) is n o t s u r p r i s i n g i n v i e w o f the p o t e n t i a l m e m b r a n e t o x i c i t y o f c o m p o u n d s capable of altering membrane structure.

Acknowledgment W e are g r a t e f u l t o M r s . U . B e h r e n d f o r c o m p e t e n t t e c h n i c a l assistance a n d to Prof. O . F e n n e m a a n d t w o u n k n o w n reviewers for critical a n d helpful comments.

O r i g i n a l r e s e a r c h r e p o r t e d i n this p a p e r w a s s u p

ported b y the Deutsche Forschungsgemeinschaft.

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Lovelock,J. E. Biochim. Biophys. Acta 1953, 10,414-426. Lovelock,J. E . Proc. R. Soc. Med. 1954,47,60-65. Jagendorf,A. T.; Avron,M. J. Biol. Chem. 1958, 231,377-290. Duane,W.; Krogmann,D. W. Biochim. Biophys. Acta 1963,71, 195-196. Heber,U.; Santarius,K. A. Plant Physiol. 1964, 39,712-719. Santarius,Κ. Α.; Heber,U. Cryobiology 1970,7,71-78. Santarius,K. A. Biochim. Biophys. Acta 1973, 291,35-50. Porter,V. S.; Denning,N. P.; Wright,R. C.; Scott,Ε. M. J. Biol. Chem. 1953,205,883-891. 9. Araki,T. Cryobiology 1977, 14, 144-150. 10. Farrant,J. Nature 1965,205,1284-1287. 11. Souzu,H. Arch. Biochem. Biophys. 1967, 120,344-351.



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12. Levitt,J. "Responses of Plants to Environmental Stresses"; Academic: New York,1972. 13. Meryman,H. T.; Williams,R. J.; Douglas,M. S. J. Cryobiology 1977, 14, 287-302. 14. Heber,U.; Santarius,K. A. "Temperature and Life"; Precht,H.,Christophersen,J.,Hensel,H. Larcher,W.,Eds.; Springer: Berlin,1973,pp 232-263. 15. Steponkus,P. L.; Garber,M. P.; Myers,S. P.; Lineberger,R. D. Cryo biology 1977, 14,303-321. 16. Asahina,E. In "Cellular Injury and Resistance in Freezing Organisms"; Asahina,E.,Ed.; Low Temp. Sci.,Ser. Β (Teion Kagaku,Seibutsu-Hen) 1967, II, 211-229. 17. Mazur,P. Fed. Proc. Am. Soc. Exp. Biol. 1965,24, 175-182. 18. Mazur,P. Science 1970, 168,939-949. 19. Mazur,P.; Leibo,S. P.; Chu,Ε. Η. J. A. Exp. Cell Res. 1972,71,345-355. 20. Santarius,Κ. Α.; Heber,U. Planta 1967,73,109-137. 21. Santarius,K. A. Plant Physiol. 1971, 48,156-162. 22. Heber,U.; Ernst,R. In "Cellular Injury and Resistance in Freezing Orga nisms"; Asahina,E.,Ed.; Low Temp. Sci.,Ser. Β (Teion Kagaku,Sei butsu-Hen) 1967,II,63-77. 23. Heber,U.; Tyankova,L.; Santarius,K. A. Biochim. Biophys. Acta 1971, 241,578-592. 24. Santarius,K. A. Planta 1969,89,23-46. 25. Farrant,J.; Woolgar,A. E. In "The Frozen Cell"; Wolstenholf,G. E . W., O'Connor,Μ.,Eds.; J. & A. Churchill: London,1970; pp 97-119. 26. Ryrie,I. J. Jagendorf,A. T. J. Biol. Chem. 1971, 246,582-588. 27. Clendenning,K. A. In "Encyclopedia of Plant Physiology"; Ruhland,W., Ed.; Springer: Heidelberg,1960; Vol. V, 1,pp 736-772. 28. Heber,U.; Tyankova,L.; Santarius,K. A. Biochim. Biophys. Acta 1973, 291, 23-37. 29. Heber, U. Cryobiology 1968,5,188-201. 30. Heber,U.; Kempfle,M. Z. Naturforsch. Teil Β 1970,25b,834-842. 31. Volger,H. G.; Heber,U. Biochim. Biophys. Acta 1975,412,335-349. 32. Santarius,Κ. Α.; Ernst,R. Planta 1967,73,91-108. 33. Santarius,K. A. Planta 1973, 113,105-114. 34. Santarius,Κ. Α.; Heber,U. (1972) Proc. Colloq. on the Winter Hardiness of Cereals,Agric. Res. Inst.,Hungarian Academy of Science,Martonvasar,pp 7-29. 35. Lovelock,J. E. Biochim. Biophys. Acta 1953, 11,28-36. 36. Rostand,J. C.R. Acad. Sci. 1946,234,2301-2312. 37. Polge,C.; Smith,Α.; Parkes,A. S. Nature (London) 1949, 164,666. 38. Meryman,Η. T. Nature (London) 1968,218,333-336. 39. Neumann,J.; Jagendorf,A. T. Arch. Biochem. Biophys. 1964,107, 109119. 40. Mitchell,P. Biol. Rev. 1966,41,445-502. 41. Heber,U. Plant Physiol. 1967,42, 1343-1350. 42. Garber,Μ. P.; Steponkus,P. L. Plant Physiol. 1976,57,673-680. 43. Packer,L.; Crofts,A. R. In "Current Topics in Bioenergetics"; Sanadi, D. R.,Ed.; Academic: New York,1967; Vol. 2,pp 24-64. 44. Crofts,A. R.; Deamer,D. W.; Packer,L. Biochim. Biophys. Acta 1967, 131,97-118. 45. Deamer,D. W.; Packer,L. Biochim. Biophys. Acta 1969, 172,539-545. 46. Karlish,S. J. D.; Shavit,N.; Avron,Μ. Eur. J. Biochem. 1969,9, 291-298. 47. Deamer,D. W.; Crofts,A. R.; Packer,L. Biochim. Biophys. Acta 1967, 13,81-96. 48. Volger,H. G.; Heber,U.; Berzborn,R. J. Biochim. Biophys. Acta 1978, 511,455-469. ;



188

PROTEINS AT LOW TEMPERATURES

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RECEIVED June 16, 1978.


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