9 The Use of Carbon-13 NMR to Study Binding of
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Hormones to M o d e l Receptor Membranes: The O p i o i d Peptide Enkephalin HAROLD C. JARRELL, P. TANCREDE, ROXANNE DESLAURIERS, and IAN C. P. SMITH Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6 W. HERBERT McGREGOR Wyeth Laboratories, P. O. Box 8299, Philadelphia, PA 19101 The recent discovery of a class of peptides, the enkephalins, which act as opiate agonists has led to a number of physical chemical studies aimed at understanding the structure-activity relationships between the enkephalins and the opiates (1-9). The H spin-spin coupling constants [2-4] of the enkephalins in solution can be interpreted in terms of folded conformations resembling that of morphine in the placement of the residues which appear important for biological activity. X-ray crystallo graphy and theoretical calculations (4-9) have also shown that methionine and leucine enkephalin adopt conformations similar to those concluded from H NMR studies. Hence i t would appear that opioid peptides can topographically resemble the opiates by assuming preferred, folded, conformations. However, earlier studies from this laboratory (10) have shown that C NMR data can be interpreted in terms of a conformationally flexible structure for methionine enkephalin. Abood and coworkers (11-13) have reported that acidic lipids, in particular phosphatidyl serine (PS), enhanced binding of opiates to membranous components of brain. Furthermore, PS has been shown to bind opiates stereospecifically whereas other acidic lipids bind to a lesser extent and neutral lipids bind very l i t t l e (1J). Loh and coworkers C!l~Lê) have reported that cerebroside *Issued as N.R.C.C. Publication No. 17068. 1
1
13
0-8412-0505-l/79/47-103-159$05.50/0 © 1979 American Chemical Society Pasika; Carbon-13 NMR in Polymer Science ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
160
CARBON-13 NMR IN POLYMER SCIENCE
s u l f a t e e x h i b i t s s t r o n g b i n d i n g o f o p i a t e s and t h a t t h e
cerebro-
s i d e s u l f a t e - o p i a t e c o m p l e x r e s e m b l e s an o p i a t e complex w h i c h had been i s o l a t e d f r o m b r a i n c o m p o n e n t s .
Lipids,
therefore,
i m p l i c a t e d i n the i n t e r a c t i o n of o p i a t e s w i t h t h e i r It
i s known
(17)
t h a t o p i a t e s and o p i o i d p e p t i d e s may
s p e c i f i c receptor binding (high a f f i n i t y ) affinity)
binding.
Phosphatidyl
Preliminary
and n o n s p e c i f i c
s e r i n e and o t h e r
weak b i n d i n g o f o p i a t e s and t h e r e f o r e specific binding.
have been
receptors.
lipids
exhibit (low
exhibit
s e r v e as m o d e l s f o r
non
to a study of high a f f i n i t y
binding
we c h o s e t o s t u d y t h e i n t e r a c t i o n o f m e t h i o n i n e e n k e p h a l i n
to
various
In
lipids,
i n p a r t i c u l a r PS,
a s a model
t h e s e s t u d i e s we have f o l l o w e d a number o f
for binding. C
13
NMR p a r a m e t e r s
t h e g l y c i n e - 2 and g l y c i n e - 3 r e s i d u e s o f m e t h i o n i n e The g l y c y l This
r e s i d u e s were 90% e n r i c h e d i n
a l l o w e d us t o measure c h e m i c a l s h i f t s
and s p i n - l a t t i c e r e l a x a t i o n t i m e s
(Tj
C
13
enkephalin.
a t the
α-carbons.
( δ ) , l i n e w i d t h s (Δν),
for
the α-carbons of
g l y c i n e r e s i d u e s as a f u n c t i o n o f v a r i o u s p a r a m e t e r s .
It
g e n e r a l l y b e l i e v e d t h a t the binding i s e l e c t r o s t a t i c i n ( V [ , l _ 5 ) ; we t h e r e f o r e
s o u g h t a pH dependence o f
e n k e p h a l i n to the l i p i d s .
We f u r t h e r
M a t e r i a l s and
of
between
methionine
( T y r G l y G l y P h e M e t ) , e n r i c h e d t o 90% i n (10).
Lipid Products,
South N u t f i e l d ,
t a i n e d from L i p i d
Products,
(sulfatides)
s u l f a t e pentahydrate
1 3
Phosphatidyl
o b t a i n e d from Serdary Research L a b o r a t o r i e s ,
Don M i l l s
the b i n d i n g
Methods
p a r e d as d e s c r i b e d p r e v i o u s l y
sulfate
nature
PS.
[ 2 - [ 2 - 1 3 C ] g l y c i n e ] and [ 3 - [ 2 - 1 3 C ] g l y c i n e ] enkephalin
the
is
sought c o m p e t i t i o n
m o r p h i n e and e n k e p h a l i n f o r b i n d i n g t o
for
England.
were
s e r i n e (PS)
l e c i t h i n was
England.
was o b t a i n e d f r o m A n a l a b s ,
Inc.
or ob
Morphine Limited,
Samples f o r b i n d i n g s t u d i e s
p r e p a r e d as d e s c r i b e d p r e v i o u s l y
was
Cerebroside
was p u r c h a s e d f r o m Ingram and B e l l
( T o r o n t o ) , Canada.
pre
L o n d o n , Canada
Egg
South N u t f i e l d ,
C,
were
(10).
NMR measurements were p e r f o r m e d on s a m p l e s p r e p a r e d
Pasika; Carbon-13 NMR in Polymer Science ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
in
9. JARRELL ET AL.
Opioid
Peptide
deuterium oxide a t 30°C,
Enkephalin
161
pH v a l u e s a r e m e t e r r e a d i n g s
n o t been c o r r e c t e d f o r t h e d e u t e r i u m
that
isotope e f f e c t .
The pH o f
s a m p l e s was v a r i e d u s i n g h y d r o c h l o r i c a c i d and s o d i u m d i l u t e d i n deuterium 1 3
C
previously
QO).
to external
1 3
hydroxide
oxide.
NMR measurements
XL-10Q s p e c t r o m e t e r s
have
were p e r f o r m e d
on V a r i a n C F T - 2 0 and
u n d e r c o n d i t i o n s w h i c h have been d e s c r i b e d C chemical s h i f t s are reported with
tetramethylsilane
s a m p l e s as an i n t e r n a l
(TMS).
standard
respect
D i o x a n e was added t o t h e
f o r l i n e w i d t h and c h e m i c a l
shift
measurements. R e s u l t s and D i s c u s s i o n Three parameters a r e r e a d i l y o b t a i n a b l e w h i c h may be u s e f u l cal
shift
i n studying
(
