Physiological Effects of Food Carbohydrates

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 4, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/bk-1975-0015.ch016

α-Amylase Inhibitors from Plants J. JOHN MARSHALL Laboratories for Biochemical Research, Howard Hughes Medical Institute and Department of Biochemistry, University of Miami School of Medicine, Miami, Fla. 33152

Introduction

Many plants contain substances which are inhibitory to enzyme action and among the most widespread of such compounds are inhibitors of hydrolytic enzymes. In this article, the nature and distribution of enzyme inhibitors in plants is reviewed, and possible in vivo functions of these inhibitors are discussed. Particular consideration will be given to the proteinaceous inhibitors of the important digestive enzyme, α-amylase, including the detection, assay, specificities and other properties, and physiological effects of these inhibitors. Distribution and Function of Hydrolytic Enzyme Inhibitors A survey of the literature, summarized in Table 1, has reveal ed reports of inhibitors of proteases, ribonuclease, invertase, polygalacturonase, cellulase and α-amylase from plant sources. These inhibitors are of two general types - those which are proteins and are rather specific for a particular enzyme, and those which are non-proteinaceous, usually polyphenolic or tannin in nature. Inhibitors of the latter type are much less specific, often inhibiting a number of quite different enzymes. Proteinaceous i n h i b i t o r s o f p r o t e o l y t i c enzymes have been i s o l a t e d from many p l a n t s , and t h e i r chemistry has been examined in d e t a i l . Primary s t r u c t u r e s o f many o f these i n h i b i t o r s a r e known, c r e d i b l e mechanisms o f a c t i o n have been p o s t u l a t e d , and the nature o f p r o t e a s e - i n h i b i t o r i n t e r a c t i o n has been examined by Xray c r y s t a l l o g r a p h y (1-4). The r o l e o f protease i n h i b i t o r s i n p l a n t s has not been e s t a b l i s h e d unequivocally; p o s s i b l e functions include t h e i r involvement i n the r e g u l a t i o n o f the a c t i v i t y o f plant proteases, p r o t e c t i o n o f p l a n t s against m i c r o b i a l a t t a c k o r insect prédation, o r that they serve as storage p r o t e i n s (3)· I n h i b i t o r s o f other h y d r o l y t i c enzymes have been examined i n much l e s s d e t a i l than the protease i n h i b i t o r s and, with the exception o f α-amylase i n h i b i t o r s , only b r i e f mention o f them w i l l 244 In Physiological Effects of Food Carbohydrates; Jeanes, Allene, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

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α-Amylase Inhibitors from Plants

TABLE 1


NATURALLY OCCURRING INHIBITORS OF HYDROLYTIC ENZYMES

Enzyme

Source o f Inhîbî t o r

Nature o f Inhibitor

References

Protease

Many p l a n t s

Protein

I- 3

Invertase

Potato Maize Ipomoea p e t a l s

Protein Protein Protein

9 10

Polygalacturonase

Bean hypocotyls, tomato stems, c u l t u r e d sycamore eel 1 s

Protein

I I - 13

Cellulase

Many p l a n t s

Polyphenol

14-17

Rîbonuclease

L i l a c leaves

Probably protein

19

or Amylase

Many p l a n t s

Polyphenol and p r o t e i n

See Table 2

5-8

In Physiological Effects of Food Carbohydrates; Jeanes, Allene, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.


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be made. Invertase i n h i b i t o r s , which are p r o t e i n s , probably serve to regulate sucrose breakdown i n p l a n t s . This can be s a i d w i t h some degree o f c e r t a i n t y because they do i n h i b i t the corresponding plant invertases (5~8). I t has been suggested (11-13) that poly galacturonase i n h i b i t o r s , which are a l s o p r o t e i n s , are involved i n p r o t e c t i n g p l a n t s against invasion by plant pathogens, which u s u a l l y gain entry i n t o p l a n t s by breakdown o f the c e l l w a l l s under the a c t i o n o f p e c t i c enzymes. C e l l u l a s e i n h i b i t o r s (14-17) are, apparently without e x c e p t i o n , polyphenolic and may play a r o l e i n p r o t e c t i n g the s t r u c t u r a l m a t e r i a l of p l a n t s against degradation by c e l l u l o l y t i c micro-organisms. It i s probable that they a l s o a c t to i n h i b i t the c e l l u l o l y t i c enzymes o f rumen micro organisms, so that such i n h i b i t o r s may have an undesirable e f f e c t on the growth c h a r a c t e r i s t i c s o f animals fed on m a t e r i a l s r i c h i n such i n h i b i t o r s (15,17,18). There i s only one report (19) o f an i n h i b i t o r o f ribonuclease i n p l a n t s . It was suggested that t h i s i n h i b i t o r i s proteinaceous; i t s in vivo f u n c t i o n i s not known. L i k e the protease i n h i b i t o r s , i n h i b i t o r s o f α-amylase appear to be widely d i s t r i b u t e d throughout the plant kingdom, and because of t h e i r a b i l i t y to i n a c t i v a t e α-amylase in vivo, they may be o f c o n s i d e r a b l e n u t r i t i o n a l s i g n i f i c a n c e . For t h i s reason, much a t t e n t i o n i s now being focussed on these i n h i b i t o r s . Their f u n c t i o n i n p l a n t s i s not y e t known with c e r t a i n t y . However, con s i d e r a t i o n o f the s p e c i f i c i t i e s o f α-amylase i n h i b i t o r s (vide infra) suggests that they might serve to p r o t e c t the seeds i n which they occur against prédation by i n s e c t s and animals, by v i r t u e o f t h e i r I n h i b i t o r y a c t i v i t y towards the d i g e s t i v e amylases o f the predators. Most α-amylase i n h i b i t o r s are i n a c t i v e towards m i c r o b i a l and plant α-amylases, i n d i c a t i n g that they are not a n t i m i c r o b i a l agents, nor do they serve t o regulate the l e v e l s o f α-amylase i n the p l a n t s . The remainder o f t h i s a r t i c l e reviews the current s t a t u s o f our knowledge regarding i n h i b i t o r s of α-amylase. Detection

and Assay of α-Amylase

Inhibitors

A v a r i e t y o f methods f o r determination o f α-amylase i n h i b i t o r a c t i v i t i e s i n p l a n t e x t r a c t s have been used, and v i r t u a l l y a l l workers have expressed i n h i b i t o r a c t i v i t i e s i n d i f f e r e n t ways. However, with only a few exceptions i t has been r e a l i z e d that i t i s necessary to pre-incubate α-amylase and i n h i b i t o r f o r i n h i b i t ion t o take place, rather than simply t o incorporate i n h i b i t o r d i r e c t l y Into a d i g e s t c o n t a i n i n g a mixture o f enzyme and s u b s t r a t e . For the most p a r t , the d u r a t i o n , pH, temperature and other c o n d i t i o n s o f pre-incubât ion appear t o have been a r b i t r a r i l y chosen. A f t e r p r e - i n c u b a t i o n , u n i n h i b i t e d amylase a c t i v i t y remaining has been determined by a v a r i e t y o f methods, most u s u a l l y based on the increase o f reducing power [measured w i t h dlnltrosalîcylic a c i d (20) o r an a l k a l i n e copper reagent ( 2 1 ) ] , o r by the decrease i n iodine s t a i n i n g power (22), during a c t i o n on



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s t a r c h . Amylase i n h i b i t o r a c t i v i t i e s have in many instances been expressed in u n i t s , based on the amount o f i n h i b i t o r which w i l l I n h i b i t a c e r t a i n p r o p o r t i o n o f a f i x e d amount o f α-amylase. I t has not u s u a l l y been made c l e a r whether the measured extent o f i n h i b i t i o n represents the maximum o b t a i n a b l e w i t h the amount o f i n h i b i t o r used, o r whether i t i s l e s s than the maximum o b t a i n a b l e w i t h that amount o f i n h i b i t o r . In the l a t t e r case, i n h i b i t o r a c t i v i t y i s being measured i n terms o f the r a t e o f amylase i n h i b i t i o n , rather than r e f l e c t i n g the s t o i c h i o m e t r y o f amylaseinhibitor interaction. During recent s t u d i e s (23) on the amylase i n h i b i t o r from kidney beans Phaseolus vulgaris, p a r t i c u l a r a t t e n t i o n was given t o the s e l e c t i o n o f pre-incubation c o n d i t i o n s which express optimal i n h i b i t o r a c t i v i t y . The f i n d i n g s h i g h l i g h t e d the importance o f c a r e f u l choice of the p r e - i n c u b a t i o n c o n d i t i o n s . Assays based on determination o f the t o t a l amount o f α-amylase which a sample o f the i n h i b i t o r can render i n a c t i v e were u n s a t i s f a c t o r y . Extended pre-incubation times were required t o achieve maximum i n h i b i t i o n , and l o s s o f α-amylase a c t i v i t y tended t o occur during the pre incubation p e r i o d , even i n the absence o f i n h i b i t o r . Measurement of the r a t e o f i n h i b i t i o n o f α-amylase was found t o be much more convenient and accurate,and required only short p r e - i n c u b a t i o n times. The c o n d i t i o n s found s u i t a b l e f o r assay o f the kidney bean i n h i b i t o r are summarized in Scheme I. A temperature o f 37°C and a pH o f 5.5 are used f o r the p r e - i n c u b a t i o n , these c o n d i t i o n s being f a v o r a b l e f o r r a p i d i n h i b i t i o n o f hog p a n c r e a t i c α-amylase by the kidney bean i n h i b i t o r (23). Calcium c h l o r i d e and human serum albumin are included i n the p r e - i n c u b a t i o n d i g e s t , t o pro t e c t the α-amylase against spontaneous i n a c t i v a t i o n . Amylase a c t i v i t y i s measured, a f t e r p r e - i n c u b a t i o n , by a d d i t i o n o f s t a r c h buffered a t pH 6.9, and determination o f the decrease i n i o d i n e s t a i n during incubation f o r 5 min. One u n i t o f i n h i b i t o r a c t i v i t y i s defined as the amount which causes 50% i n h i b i t i o n o f the aamylase during p r e - i n c u b a t i o n f o r 20 min. The extent o f i n h i b i t ion i s p r o p o r t i o n a l t o the amount o f i n h i b i t o r present and t o the d u r a t i o n o f p r e - i n c u b a t i o n , provided the extent o f incubation does not exceed 50%. Thus the amount o f i n h i b i t o r and the d u r a t i o n o f pre-incubation must be such as t o g i v e no more than t h i s degree o f i n h i b i t i o n in order t o ensure a v a l i d assay. A simple method f o r d e t e c t i o n o f amylase i n h i b i t o r s i n e x t r a c t s o f b i o l o g i c a l m a t e r i a l s has r e c e n t l y been described (24). A narrow c e l l u l o s e s t r i p saturated w i t h the s o l u t i o n being exam ined f o r i n h i b i t o r , i s placed on a buffered starch-agar gel p l a t e for 2 hours a t 37°C. The s t r i p i s then removed and another narrow s t r i p saturated w i t h amylase s o l u t i o n i s placed on the gel a t r i g h t angles across the p o s i t i o n occupied by the f i r s t s t r i p . A f t e r incubation f o r 6-18 hr a t 37°C., the second s t r i p i s removed and the s l a b i s flooded w i t h i o d i n e s o l u t i o n , amylase a c t i v i t y being shown by c l e a r zones on a purple background. The presence


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SCHEME I ASSAY OF Q.-AMYLASE INHIBITOR ACTIVITY (23) Ppe-inoubation:

A 1.5 ml d i g e s t c o n t a i n i n g


and

hog pancreatic α-amylase (1 yg) calcium c h l o r i d e (1.5 mg) human serum albumin (1.5 mg) acetate b u f f e r 6.7 mM, pH 5-5) inhibitor

i s incubated a t 37°C f o r a s u i t a b l e period o f time ( u s u a l l y 20 min). The amount o f i n h i b i t o r present should be s u f f i c i e n t t o cause 10-50? i n h i b i t i o n of the α-amylase during the pre-incubation period. Assay of Residual ^.-Amylase Activity

Controls:

:

Buffered s o l u b l e s t a r c h s o l u t i o n (1.0 ml c o n t a i n i n g 10 mg s t a r c h i n 100 mM glycerophosphate b u f f e r pH 6.9 and 20 mM calcium c h l o r i d e ) i s added to the pre incubation mixture. The r e s u l t i n g d i g e s t i s then incubated a t 37°C f o r e x a c t l y 5 min. The absorbance o f the s t a r c h - i o d i n e complex obtained on a d d i t ion o f a sample o f the d i g e s t (0.1 ml) to 5.0 ml o f 0.02? iodine - 0.2? potassium iodide s o l u t i o n , i s measured at 680 nm. α-Amylase a c t i v i t y i s expressed as the d i f f e r e n c e i n the absorbance from that o f a substrate blank d i g e s t c o n t a i n i n g everything ex cept α-amylase. (1) α-Amylase pre-incubated without i n h i b i t o r (gives the u n i n h i b i t e d a c t i v i t y ) . (2) In cases where the i n h i b i t o r preparation a l s o contains amylase a c t i v i t y , a sample of the i n h i b i t o r preparation i s preincubated w i t h a l l the other d i g e s t c o n s t i t u e n t s except α-amylase, then incubated w i t h s t a r c h f o r the usual time. This c o n t r o l i s u s u a l l y necessary when crude e x t r a c t s are being assayed.

Unit of Inhibitor:

1 Unit o f i n h i b i t o r i s the amount which causes 50? i n h i b i t i o n o f α-amylase i n 20 min under the above c o n d i t i o n s .


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ct-Amyhse Inhibitors from Fiants

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o f i n h i b i t o r i s i n d i c a t e d by i n t e r r u p t i o n o f the l y s i s zone where the i n h i b i t o r - c o n t a i n i n g and amylase-containing s t r i p s crossed. A v a r i a t i o n o f the method, i n v o l v i n g measurement o f the diameter of c l e a r e d zones produced by a m y l a s e - i n h i b i t o r mixtures placed i n s ^ a l l w e l l s cut i n the starch-agar gel s l a b , was a l s o d e s c r i b e d . \x\ view o f the extended time required t o o b t a i n r e s u l t s , and t h e i r e s s e n t i a l l y q u a l i t a t i v e nature, there appears t o be l i t t l e advant age gained by the use o f t h i s method, rather than a more conventtonal i n h i b i t o r assay, such as that described above. Properties

of ^Amylase

Inhibitors

General Properties. α-Amylase i n h i b i t o r s have been known f o r a c o n s i d e r a b l e time, but compared with the protease i n h i b i t o r s , l i t t l e i s understood about t h e i r s t r u c t u r e s and mechanism o f a c t i o n , t h e i r n u t r i t i o n a l s i g n i f i c a n c e , o r t h e i r r o l e in vivo. Proteinaceous and ηοη-proteînaceous i n h i b i t o r s o f α-amylase have been discovered (Table 2). Insoluble α-amylase i n h i b i t o r s , such as the "sisto-amylase" described by Chrzaszcz and J a n i c k i (30,31) may f u n c t i o n simply by adsorption o f the enzyme, preventing i t s i n t e r a c t i o n w i t h sub s t r a t e , rather than by formation o f a s p e c i f i c enzyme-inhibitor complex. An e t h e r - s o l u b l e substance from navy beans (54) i n t e r f e r e s w i t h α-amylase a c t i o n on s t a r c h probably by i n t e r a c t i n g w i t h the s u b s t r a t e r a t h e r than the enzyme. Neither o f these substances can be considered a true i n h i b i t o r o f α-amylase. Potato tubers and c e r t a i n v a r i e t i e s o f sorghum contain d i a l y z a b l e , nonproteinaceous, α-amylase i n h i b i t o r s (25-28). The i n h i b i t o r from sorghum i s the b e t t e r c h a r a c t e r i z e d ; i t i s d i a l y z a b l e , heats t a b l e (even on a u t o c l a v i n g ) , and o r g a n i c - s o l v e n t s o l u b l e (2 7). It i n h i b i t s many enzymes besides α-amylase, and appears t o be a tannin o f the leucocyanidin group (28). The p r o p e r t i e s o f the i n h i b i t o r from sorghum c o n t r a s t w i t h those o f the proteinaceous α-amylase i n h i b i t o r s , which a r e s p e c i f i c f o r α-amylase, a r e n o n - d i a l y z a b l e , and may be i n a c t i v a t e d by heating. Proteinaceous α-amylase i n h i b i t o r s , although suscept i b l e t o heat i n a c t i v a t i o n , a r e g e n e r a l l y rather h e a t - s t a b l e p r o t e i n s , o f t e n r e q u i r i n g prolonged heating a t elevated temperat ures, o r even a u t o c l a v i n g , t o destroy t h e i r a c t i v i t y completely. Of a l l the proteinaceous α-amylase i n h i b i t o r s which have been i s o l a t e d from p l a n t s (Table 2), the best known i s that from wheat, f i r s t studied by Kneen and h i s co-workers (36,37) i n the 1940's, although the i n h i b i t o r s i n rye (36,37), and i n navy beans (51) were f i r s t detected a t about the same time. Only those α-amylase i n h i b i t o r s o f the proteinaceous type w i l l be considered i n d e t a i l . In the f o l l o w i n g s e c t i o n s the s p e c i f i c i t i e s o f i n h i b i t o r s from d i f f e r e n t sources w i l l be con s i d e r e d , then the p r o p e r t i e s o f the two b e s t - c h a r a c t e r i z e d proteinaceous i n h i b i t o r s o f α-amylase, those from wheat and kidney beans, w i l l be d i s c u s s e d .


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TABLE 2


NATURALLY OCCURRING INHIBITORS OF a-AMYLASE

Source o f inhibitor

Nature o f Inhibitor

Potato tubers

Characteristics

References

Non-proteinaceous

Ether-soluble; dialyzable

25,26

Sorghum

Tannins o f the leucocyanidin group

Organic-solvent soluble; general enzyme i n a c t i v a t o r

27,28

Acorn

Not

Also inhibited 3-amylase

29

Buckwheat mal t

Protein

Water-insoluble; h e a t - l a b i l e (500C)

30,31

Oat seeds

Protein

Non-dialyzable; h e a t - s t a b l e (100°C); also inhibited 3-amylase; i n h i b i t i o n reversed by s u l f h y d r y l compounds

32

Mangoes

Protein

Heat-labile (unspecî f i e d temperature); non-dialyzable

33

Colooasia esculenta tubers

Protein

Heat-stable (100°C); destroyed by autoclaving; non-dialyzable

34,35

Wheat

Protein

Refer t o t e x t

36-49

Rye

Protein

Heat-stable (100°C); non-dialyzable

45,50

Phaseolus vulgaris

Protein

Refer t o t e x t

23,51-53

identified



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Specificities of α-Amylase Inhibitors. Of the α-amylase I n h i b i t o r s which are protelnaceous In nature, only two have been reported t o i n h i b i t enzymes other than α-amylase. An i n h i b i t o r preparation from navy beans a l s o i n h i b i t e d t r y p s i n ( 5 1 ) , but the a c t i v i t y towards the l a t t e r enzyme was probably due t o contamin a t i o n by a s p e c i f i c t r y p s i n i n h i b i t o r , a l s o known t o be present In these beans ( 5 5 ) . An I n h i b i t o r preparation from oats (32) i n h i b i t s both β-amylase and α-amylase. The a b i l i t y t o r e s t o r e the a c t i v i t y of these enzymes by a d d i t i o n o f s u l f h y d r y l reagents, such as g l u t a t h i o n e , i n d i c a t e d that the i n h i b i t i o n was the r e s u l t o f I n t e r a c t i o n o f the polypeptide i n h i b i t o r w i t h t h i o l groupings i n the enzymes, rather than a s p e c i f i c p r o t e i n - p r o t e i n i n t e r a c t i o n as i s considered t o take place w i t h other i n h i b i t o r s . Thus the I n h i b i t o r from oat seeds, although apparently protelnaceous, belongs t o a d i f f e r e n t c l a s s from the s p e c i f i c proteinaceous α-amylase i n h i b i t o r s , In which we are p r i m a r i l y Interested In t h i s a r t i c l e . α-Amylases from d i f f e r e n t sources show d i f f e r e n c e s i n s u s c e p t i b i l i t y t o i n h i b i t i o n by the s p e c i f i c proteinaceous i n h i b i t ors o f α-amylase (Table 3 ) . Furthermore, these i n h i b i t o r s do not a l l have the same s p e c i f i c i t y . The I n h i b i t o r from Colocasia esculenta ( 3 4 , 3 5 ) has the simplest s p e c i f i c i t y ; s a l i v a r y aamylase i s the only enzyme i t has been found t o i n h i b i t . Kneen gnd Sandstedt reported that the wheat i n h i b i t o r was a c t i v e towards s a l i v a r y and p a n c r e a t i c α-amylases, and a l s o the s a c c h a r i f y i n g type (56) o f b a c t e r i a l α-amylase; i t was without a c t i o n on the more common b a c t e r i a l α-amylase, the l i q u e f y i n g type, o r on p l a n t α-amylase ( 3 6 , 3 7 ) . In a more recent study o f the wheat i n h i b i t o r ( 4 0 , 4 2 ) , Shainkln and B i r k separated two p r o t e i n s w i t h I n h i b i t o r y a c t i o n . One o f these (designated Aml ) had s p e c i f i c i t y s i m i l a r t o that reported by Kneen and Sandstedt f 3 6 , 3 7 ) and was a l s o found to i n h i b i t Insect (Tenebrio molitor l a r v a l midgut) α-amylase ( 5 7 ) . The second form (Amlj) i n h i b i t e d only the i n s e c t α-amylase. S i m i l a r f i n d i n g s were reported by S i l a n o and co-workers ( 4 7 ) . The α-amylase i n h i b i t o r from rye ( 3 6 , 3 7 , 4 5 ) i n h i b i t s s a l i v a r y and p a n c r e a t i c α-amylases and, l i k e that from wheat, Is without a c t i o n on b a c t e r i a l l i q u e f y i n g α-amylase and plant α-amylase ( 5 0 ) . Jaffé and co-workers reported that a p a r t l y p u r i f i e d i n h i b i t o r from kidney beans (Phaseolus vulgaris) was a c t i v e towards s a l i v a r y and p a n c r e a t i c α-amylases, and a l s o t o a small extent against b a c t e r i a l l i q u e f y i n g α-amylase ( 5 3 ) . Studies i n our Laboratory (23) d i d not support the suggestion that b a c t e r i a l α-amylase i s i n h i b i t e d by the Phaseolus vulgaris i n h i b i t o r . The most l i k e l y explanation o f these d i f f e r e n t f i n d i n g s Is that the Venezuelan workers may not have Included adequate c o n t r o l s , t o c o r r e c t f o r the spontaneous l o s s o f α-amylase a c t i v i t y during p r e - i n c u b a t i o n . In the case o f the b a c t e r i a l enzyme, such losses are p a r t i c u l a r l y large because o f the presence o f traces o f p r o t e o l y t i c enzymes, even i n c r y s t a l l i n e preparations o f the amylase ( 5 8 ) . The i n h i b i t o r from kidney beans a l s o i n h i b i t s Helix pomatia ( s n a i l ) α-amylase, but a t about only h a l f the rate i t i n h i b i t s s a l i v a r y 2

9



+ +

+ +

+

ND

ND ND

+

ND

ND

ND

ND +

ND ND ND

.ND

ND

ND ND

ND + +

ND

ND

Insect {Tenebrio molitor)

a

s i m i l a r r e s u l t s reported i n Refs. 47 and 48.

+ indicates i n h i b i t i o n ; - i n d i c a t e s no i n h i b i t i o n ; ND i n d i c a t e s not determined. The r e l a t i v e a f f i n i t i e s o f a p a r t i c u l a r i n h i b i t o r f o r the d i f f e r e n t α-amylases which i t i n h i b i t s , when these a r e known, are discussed i n the t e x t .

52,53 23

ND

-

-

+

+

Phaseolus vulgaris

a

-

42

+

36,37

Wheat Ami j

-

-

+

+

-

-

-

-

50

Rye

α-Amylase Plant Helix Bacterial Bacterial pomatia (1iquefying) (sacchari f y i n g )

Fungal

Pancreatic

+

34,35

Colooasia esculenta

Salivary

OF INHIBITORS TOWARDS a-AMYLASES FROM DIFFERENT SOURCES

Ami 2

Reference

Source o f Inhibitor

SPECIFICITIES

TABLE 3



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253

and p a n c r e a t i c α-amylases, the l a t t e r two enzymes beinq i n a c t i v a t e d a t almost i d e n t i c a l rates ( 2 3 ) . In t h i s respect the bean i n h i b i t o r d i f f e r s markedly from one cereal (rye) α-amylase i n h i b i t o r , which acts on the s a l i v a r y enzyme about 10-times f a s t e r than on the p a n c r e a t i c enzyme ( 5 0 ) . One p o s s i b l e e x p l a n a t i o n f o r the d i f f e r e n c e i n rate of i n h i b i t i o n of these two α-amylases i s that the carbohydrate present i n the l a t t e r ( 5 9 ) , but apparently absent from the former, i n t e r f e r e s w i t h enzyme-inhibitor i n t e r a c t i o n . Decrease in s u s c e p t i b i l i t y to i n h i b i t i o n by n a t u r a l l y o c c u r r i n g proteinaceous i n h i b i t o r s has p r e v i o u s l y been observed a f t e r chemical attachment of carbohydrate to p a n c r e a t i c α-amylase and p a n c r e a t i c t r y p s i n (60). At the present time, the d i f f e r e n c e s in s u s c e p t i b i l i t y of amylases to i n h i b i t i o n are not c l e a r l y under stood. However the p o s s i b i l i t y does e x i s t that s t u d i e s o f enzymei n h i b i t o r i n t e r a c t i o n w i l l e v e n t u a l l y be of value in examinations of the s t r u c t u r e s and a c t i v e s i t e topography of α-amylases. α-Amylase Inhibitors from Wheat. The α-amylase i n h i b i t o r from wheat has received more a t t e n t i o n than any of the other aamylase i n h i b i t o r s . I n i t i a l s t u d i e s on the i n h i b i t o r ( 3 6 , 3 7 ) , prepared by e x t r a c t i o n of wheat f l o u r w i t h water, followed by b o i l i n g to destroy endogenous amylase a c t i v i t y , showed i t to have the p r o p e r t i e s of a p r o t e i n . Thus the i n h i b i t o r was p r e c i p i t a t e d by ammonium s u l f a t e , and by high concentrations of e t h a n o l . Although i t was q u i t e thermostable, being l i t t l e a f f e c t e d by prolonged storage at 70°C., or on b o i l i n g f o r short p e r i o d s , autoc l a v i n g at 121°C f o r 30 min caused complete l o s s of i n h i b i t o r a c t i v i t y . As w e l l as being w a t e r - s o l u b l e , the i n h i b i t o r was a l s o s o l u b l e in aqueous ethanol at concentrations up to 70? e t h a n o l . The ethanol s o l u b i l i t y was used to demonstrate that amylase i n h i b i t i o n was r e v e r s i b l e , a d d i t i o n of ethanol to a f i n a l c o n c e n t r a t i o n of 70? p r e c i p i t a t i n g α-amylase from a mixture of amylase and i n h i b i t o r , l e a v i n g the i n h i b i t o r i n s o l u t i o n . Kneen and Sandstedt ( 3 6 , 3 7 ) reported that complete i n h i b i t i o n of aamylase w i t h the wheat i n h i b i t o r could not be achieved; this observation may i n d i c a t e that amylase i n h i b i t i o n i s p a r t l y r e v e r s i b l e by s u b s t r a t e . Treatment w i t h pepsin rendered the inhibitor inactive. The production of wheat i n h i b i t o r w i t h kernel development was f o l l o w e d , and showed that the i n h i b i t o r was produced j u s t as the kernels reached m a t u r i t y , approximately 15 days a f t e r f l o w e r i n g (36,37). It d i d not disappear on germination. Since the l e v e l of i n h i b i t o r was markedly lower in wheat brans than in whole ground wheat, the suggestion was made that the m a j o r i t y of the i n h i b i t o r i s located in the endosperm. The i n h i b i t o r was present i n s i m i l a r amounts i n a l l wheats t e s t e d , namely hard red w i n t e r wheat, s o f t red w i n t e r wheat, and hard red s p r i n g wheat. Subsequent work (38) on the wheat i n h i b i t o r r e s u l t e d i n a 7 5 0 - f o l d p u r i f i c a t i o n from wheat e x t r a c t s by a 70°C heat treatment, ethanol f r a c t i o n a t i o n and adsorption on alumina. The a b i l i t y to



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i n a c t i v a t e the i n h i b i t o r by t r e a t i n g with n i t r o u s a c i d , which reacts with p r o t e i n amino groups, and by d i g e s t i o n with the p r o t e o l y t i c enzyme, f i c i n , was presented as f u r t h e r evidence that the i n h i b i t o r i s a p r o t e i n . Papain, however, did not destroy i t s a c t i v i t y . These l a t e r s t u d i e s (39) showed some d i f f e r e n c e s from the e a r l i e r f i n d i n g s (36,37). Thus, p u r i f i c a t i o n involved p r e c i p i t a t i o n with 70% e t h a n o l ; p r e v i o u s l y the i n h i b i t o r was reported to be s o l u b l e in t h i s concentration of a l c o h o l , and no reason was suggested f o r the apparent discrepancy. A f u r t h e r d i f f e r e n c e was in the e f f e c t of d i a l y s i s on the a c t i v i t y of the i n h i b i t o r . While a c t i v i t y was retained on d i a l y s i s against tap water or s a l t s o l u t i o n s , the a c t i v i t y of the p u r i f i e d material was l o s t on d i a l y s i s against d i s t i l l e d water. This observation was taken to i n d i c a t e the requirement f o r a d i a l y z a b l e inorganic c o f a c t o r f o r a c t i v i t y . The less-pure preparation used e a r l i e r d i d not lose a c t i v i t y on d i a l y s i s against d i s t i l l e d water, and i t was suggested that contaminating p r o t e i n s in that preparation in some way prevented exhaustion of c o f a c t o r during d i a l y s i s . The heat s t a b i l i t y p r o p e r t i e s of the p u r i f i e d i n h i b i t o r preparation were examined under a v a r i e t y of c o n d i t i o n s , from which i t was found that a c t i v i t y was l o s t f a s t e r in a l k a l i n e than in a c i d i c s o l u t i o n , at any p a r t i c u l a r temperature (38). Denaturation was achieved in 10 min at 95 C and pH 9.0. At pH 5.3, more than 60 min was r e q u i r e d . The s t a b i l i t y of the i n h i b i t o r , even in a c i d i c s o l u t i o n , appeared to be lower than that reported p r e v i o u s l y (36,37), but again the reason may have been the s t a b i l i z i n g e f f e c t of contaminating p r o t e i n s in the l e s s pure préparât ion. The i n h i b i t o r was i n a c t i v a t e d by a number of reducing agents, i.e. sodium s u l f i t e , hydrogen s u l f i d e and a s c o r b i c a c i d (39). O x i d i z i n g agents - c h l o r i n e , bromine, sodium c h l o r i t e and hydrogen peroxide - were even more e f f e c t i v e in causing loss of a c t i v i t y . A strong p o s i t i v e t e s t f o r tryptophan in the i n h i b i t o r p r e p a r a t i o n , together with the knowledge that o x i d i z i n g agents r e a d i l y a t t a c k tryptophan, led to the suggestion that tryptophan plays an important part in the a c t i v i t y of the i n h i b i t o r (39). Further support f o r the important r o l e of tryptophan came from the observation that aldehydes, such as acetaldehyde and benzaldehyde, which react w i t h indole r i n g s , a l s o caused i n a c t i v a t i o n . Freshly prepared s o l u t i o n s of the p u r i f i e d i n h i b i t o r increased in i n h i b i t o r y power on standing, t h i s being explained on the basis of d i s s o c i a t i o n to reveal the a c t i v e s i t e s responsible f o r combination w i t h α-amylase (39). It was a l s o shown that i n h i b i t ion of α-amylase d i d not take place instantaneously on a d d i t i o n of i n h i b i t o r , but increased during a period of pre-incubation of enzyme and i n h i b i t o r , before a d d i t i o n of s t a r c h . A study of the nature of the I n h i b i t i o n showed t h i s to be non-competitive. This l a t t e r f i n d i n g was considered to be in accord w i t h the d i f f e r e n c e In the e f f e c t of the i n h i b i t o r towards d i f f e r e n t α-amylases (vide supra). If the i n h i b i t o r acted by competing w i t h s t a r c h f o r the



16.

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MARSHALL

a c t i v e s i t e o f the enzyme, a l l amylases would have been expected to be a f f e c t e d . Since the r e a c t i o n i s non-competitive, i t f o l l o w s that c e r t a i n amylases have groupings which can combine w i t h the i n h i b i t o r , w h i l e others do not, although a l l the amylases have the c a t a l y t i c groupings necessary f o r s t a r c h h y d r o l y s i s . The i n h i b i t o r from wheat received l i t t l e f u r t h e r a t t e n t i o n u n t i l r e c e n t l y ; i t i s now being i n v e s t i g a t e d by several groups o f workers. Shainkin and B i r k (40-42) p u r i f i e d two forms o f the aamylase i n h i b i t o r from wheat f l o u r by ammonium s u l f a t e f r a c t i o n a t i o n , and chromatography on DEAE-cel1ulose and CM-cel1ulose. These two i n h i b i t o r s , designated Ami] and A m l 2 , d i f f e r e d i n s p e c i f i c i t y (Table 3 ) , i n molecular weight (18,000 f o r Ami] and 26,000 f o r A m l ) , and i n many other p r o p e r t i e s (42). D i l u t e s o l u t i o n s o f Ami] were not a f f e c t e d when b o i l e d (at u n s p e c i f i e d pH) f o r 10 min, whereas A m l l o s t 10-15? o f i t s a c t i v i t y a f t e r 1 min, and 70-80? a f t e r 10 min. Treatment w i t h 6.4 M urea f o r 24 hr had no a f f e c t on the i n h i b i t o r y a c t i v i t y o f Ami] and A m l , but reduction w i t h mercaptoethanol i n the presence o r absence o f 6.4 M urea r e s u l t e d i n t o t a l loss o f a c t i v i t y i n both cases. The d i f f e r e n c e s between the two i n h i b i t o r s were apparent from t h e i r s u s c e p t i b i l i t y t o d i g e s t i o n w i t h proteases. Ami] was i n a c t i v a t e d by t r y p s i n and chymotrypsin, whereas A m l was r e s i s t a n t t o these enzymes. Pronase completely destroyed Ami] and 60? o f the Aml£ in 3 hr; pepsin r e a d i l y i n a c t i v a t e d both forms o f the i n h i b i t o r . Ami] and A m l a l s o reacted i n a d i f f e r e n t manner when subjected t o chemical m o d i f i c a t i o n s such as e s t e r i f i c a t i o n w i t h methanol/HCl, and on carboxymethylation a t d i f f e r e n t pH values a f f e c t i n g s e l e c t i v e l y methionine, h i s t i d i n e and aromatic amino a c i d s , as w e l l as on cleavage w i t h cyanogen bromide (42). On the whole, Aml2 was more s e n s i t i v e t o these treatments than was Ami]. Cyanogen bromide treatment, performed i n a c o n t r o l l e d manner, removed a l l the a c t i v i t y o f Am12 towards s a l i v a r y α-amylase, but had l i t t l e e f f e c t on the a c t i v i t y o f A m l , o r the more s p e c i f i c i n h i b i t o r Ami], towards Tenebrio molitor α-amylase. On the basis o f t h i s f i n d i n g i t was suggested that Aml2 i s comprized o f Ami] plus an a d d i t i o n a l peptide fragment which c o n t a i n s the b i n d i n g s i t e f o r s a l i v a r y α-amylase. I n h i b i t i o n o f α-amylase by the wheat i n h i b i t o r s was markedly greater a f t e r pre-incubât ion o f enzyme w i t h i n h i b i t o r , followed by a d d i t i o n o f s u b s t r a t e , than a f t e r p r e - i n c u b a t i o n o f s u b s t r a t e w i t h i n h i b i t o r , followed by a d d i t i o n o f enzyme (41,42). A s i m i l a r s i t u a t i o n i s observed i n the case o f α-amylase i n h i b i t o r from kidney beans (vide infra). Although a d d i t i o n o f s u b s t r a t e cannot reverse i n h i b i t i o n , when α-amylase i s added t o a mixture of kidney bean i n h i b i t o r and excess s u b s t r a t e , the enzyme a c t s a t the same rate as i t does i n the absence o f i n h i b i t o r (23). Shainkin and B i r k suggested that pre-incubât ion o f s u b s t r a t e and i n h i b i t o r gives a complex i n which the amylase b i n d i n g s i t e i n the i n h i b i t o r i s masked. However, i n the absence o f any good evidence t o show that i n h i b i t o r and s u b s t r a t e do a c t u a l l y 2

2

2

2

2

2



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a s s o c i a t e , the low extent o f i n h i b i t i o n observed when α-amylase is added l a s t can be explained simply on the basis o f the a f f i n i t y o f the enzyme f o r i t s s u b s t r a t e , and the high concentration o f the s u b s t r a t e compared with i n h i b i t o r i n the mixture, so that sub s t r a t e p r o t e c t s the enzyme from i n a c t i v a t i o n by the i n h i b i t o r . The data on s o l u b i l i t y , charge p r o p e r t i e s , amino a c i d composition and molecular weights o f the i n h i b i t o r s (42) suggested a resemblance o f these p r o t e i n s to the g l i a d i n s (43) > wheat p r o t e i n s without any p r e v i o u s l y known b i o l o g i c a l a c t i v i t y . The p o s s i b l e r e l a t i o n between wheat α-amylase i n h i b i t o r and the wheat g l i a d i n s was a l s o recognized by Strumeyer, who prepared the i n h i b i t o r by water e x t r a c t i o n o f wheat f l o u r , followed by heat treatment a t 70°C., then ammonium s u l f a t e and ethanol p r e c i p i t a t i o n C45). M o l e c u l a r - s i e v e chromatography gave a value o f 55,000 f o r the molecular weight o f the i n h i b i t o r (46), t h i s being rather d i f f e r e n t from that reported by Shainkin and B i r k (42), and others (47-49). Recent s t u d i e s i n t h i s Laboratory (50), however, support Strumeyer's value. Disc e l e c t r o p h o r e s i s o f the p u r i f i e d i n h i b i t o r showed two major p r o t e i n bands, w i i c h corresponded i n e l e c t r o p h o r e t i c m o b i l i t y t o the α-glîadîns (46). The amino a c i d composition o f the i n h i b i t o r preparation showed that i t contained high amounts o f glutamine (30%) and p r o l i n e (50%) but a low l e v e l of l y s i n e ( l e s s than ] % ) . None o f these f i g u r e s i s s i m i l a r t o those i n the amino a c i d a n a l y s i s o f the two forms o f i n h i b i t o r i s o l a t e d by Shainkin and B i r k . Strumeyer and F i s h e r demonstrated complex formation between i n h i b i t o r and α-amylase by gel f i l t r a t i o n (46). S i l a n o and co-workers (47), l i k e Shainkin and B i r k (42), have separated two types o f α-amylase i n h i b i t o r from wheat, by using chromatography on columns o f Sephadex G-100. They have a l s o shown (48) that albumins from the kernels o f hexaploid wheat can be separated e l e c t r o p h o r e t i c a l l y i n t o s i x f r a c t i o n s . Five o f these albumins had very s i m i l a r p r o p e r t i e s , and were considered t o belong to a f a m i l y o f c l o s e l y r e l a t e d p r o t e i n s ; a l l i n h i b i t e d Tenebrio molitor α-amylase, but no other α-amylase. The s i x t h albumin, designated 0.19, was d i s t i n c t from the o t h e r s , and i n h i b i t e d s a l i v a r y and p a n c r e a t i c α-amylases i n a d d i t i o n t o Tenebvio molitor α-amylase. The 0.19 albumin contained two noni d e n t i c a l subunits (48). One subunît, responsible f o r the i n h i b i t o r y a c t i v i t y towards Tenebrio molitor α-amylase, was considered to be a member o f the f i v e albumin f a m i l y s p e c i f i c f o r t h i s amylase. The nature o f the second subunît, responsible f o r the i n h i b i t o r y a c t i v i t y o f 0.19 albumin towards s a l i v a r y α-amylase was not c l e a r . It was a l s o proposed (48) that 0.19 albumin c o r r e s ponded t o the Am12 i n h i b i t o r i s o l a t e d by Shainkin and B i r k (42). While these l a t t e r workers had suggested (42) that the polypeptide chain o f Aml2 c o n s i s t e d o f Ami], plus an a d d i t i o n a l peptide segment, S i l a n o and co-workers proposed (48) that Ami2, l i k e 0.19 albumin, contained two s u b u n i t s , one o f which was Aml|. The s i t u a t i o n regarding the number o f α-amylase i n h i b i t o r s i n 9



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α-Amylase Inhibitors from Pfonts

257

wheat, and t h e i r r e l a t i o n s h i p , has become more confused f o l l o w i n g a recent report from Saunders and Lang (49). These workers separated two albumin p r o t e i n s w i t h I n h i b i t o r y a c t i v i t y , termed i n h i b i t o r s I and 11, by chromatography o f a heat-treated wheat e x t r a c t on DEAE-Sephadex. I n h i b i t o r s I and II had almost i d e n t i c al p h y s i c a l p r o p e r t i e s ( pi 6.7, molecular weight 20,000 f o r I; pi 6.5, molecular weight 21,000 f o r 11) and i t i s not c l e a r whether they a r e d i s t i n c t p r o t e i n s , o r whether the f r a c t i o n a t i o n was a r t e f a c t u a l . I n h i b i t o r s I and II were d i s t i n c t from S i l a n o ' s 0J9 albumin (pi 7.3, molecular weight 23,800). Each i n h i b i t o r , I, U and 0.19 was reported t o a c t i n an uncompetitive f a s h i o n against p a n c r e a t i c α-amylase, w i t h Kj 5 x 10"8m. Further work w i l l be necessary t o c l a r i f y the exact number o f α-amylase i n h i b i t o r s present i n wheat, and t o d e f i n e t h e i r r e l a t i o n s h i p a t the molecular l e v e l . (x-Amylase Inhibitor from Phaseolus v u l g a r i s . Although I t was f i r s t detected i n the 1940's (51), the α-amylase i n h i b i t o r present fn beans d i d not r e c e i v e f u r t h e r a t t e n t i o n u n t i l r e c e n t l y . The observation that r a t s fed on d i e t s o f raw kidney beans excreted undigested s t a r c h (61) prompted Jaffé and h i s co-workers t o examine e x t r a c t s o f these beans f o r the f a c t o r s r e s p o n s i b l e . They p u r i f i e d an α-amylase i n h i b i t o r 3-fold from a crude e x t r a c t , by heat treatment a t 60°C f o r 30 min (52). The p a r t l y p u r i f i e d i n h i b i t o r , which was n o n - d l a l y z a b l e and was destroyed by heating at 100°C f o r 15 min, had the p r o p e r t i e s o f a p r o t e i n . I t s molecular weight was judged, by molecular-sieve chromatography on Sephadex, t o be greater than 50,000. The nature o f the i n h i b i t i o n was reported t o be non-competItive. I n h i b i t o r s w i t h apparently s i m i l a r p r o p e r t i e s are present i n many v a r i e t i e s o f beans and other legumes (53)· Kidney bean α-amylase i n h i b i t o r has r e c e n t l y been prepared in a high s t a t e o f p u r i t y i n t h i s Laboratory (23), by using a s e r i e s o f conventional f r a c t i o n a t i o n techniques (summarized i n Table 4). The c l o s e c o r r e l a t i o n between p r o t e i n and i n h i b i t o r a c t i v i t y In the f r a c t i o n from the CM-cellulose column ( F i g . 1), the f i n a l step i n the p u r i f i c a t i o n scheme, was taken as i n d i c a t i v e of p u r i f i c a t i o n t o homogeneity. This was confirmed by p o l y acrylamide gel e l e c t r o p h o r e s i s and a n a l y t i c a l u l t r a c e n t r i f u g a t l o n . P u r i f i c a t i o n can a l s o be achieved (23) by t a k i n g advantage o f the a f f i n i t y o f the enzyme f o r Sepharose-bound α-amylase, the i n h i b i t o r being recovered by washing w i t h pH 3.0 b u f f e r - probably as a r e s u l t o f d e s t r u c t i o n o f the a c i d - l a b i l e α-amylase. This method i s , however, not used r o u t i n e l y because p u r i f i c a t i o n can be achieved so r e a d i l y by the conventional f r a c t i o n a t i o n tech niques. Examination o f the p r o p e r t i e s o f the i n h i b i t o r (23) showed that the i n h i b i t i o n was pH dependent - a phenomenon which has not been observed, nor apparently even i n v e s t i g a t e d , i n the case o f s t u d i e s on other α-amylase i n h i b i t o r s . The maximum r a t e o f



770

142,500

150,100

116,700

Step 4 : DEAE-cellulose chromatography

Step 5 : Sephadex G-100 chromatography

Step 6 : CM-cellulose chromatography

A c t i v i t i e s determined as i n Scheme I.

1,150

203,200

Step 3 : d i a l y s i s

a

34,000

222,600

Step 2 : heat treatment (70°C., 15 min)

3,100

10,100

64,600

Total p r o t e i n (mg)

312,800

Total u n i t s

152

131

46

20.1

6.5

4.8

Specific activity (units/mg)

OF PHASEOLUS VULGARIS OL-AMYLASE INHIBITOR

Step 1 : crude e x t r a c t (500g beans)

Steps and procedures

PURIFICATION

TABLE 4

37

48

46

65

71

100

Recovery (%)

(23)


31.7

27.3

9.6

4.2

1.4

Purification


16.

MARSHALL

259

α-Amylase Inhibitors from Fiants

i n h i b i t i o n o f α-amylase i s observed a t pH 5-5, the rate dropping o f f sharply a t higher pH values. I n h i b i t o r a c t i v i t y i s a l s o s t r o n g l y dependent on the temperature o f p r e - i n c u b a t i o n ; inhibit ion o f α-amylase takes place about 20-times f a s t e r a t 37°C than a t 25°C., and i s n e g l i g i b l e a t 0°C. However, once i n h i b i t i o n has occurred, i t cannot be reversed by c o n d i t i o n s which a r e themselves unfavorable f o r i n h i b i t o r a c t i v i t y - low temperature o r a l k a l i n e pH. Neither can substrate reverse the i n h i b i t i o n , showing that aamylase has g r e a t e r a f f i n i t y f o r the i n h i b i t o r than i t has f o r i t s s u b s t r a t e . I n t e r a c t i o n o f α-amylase and i n h i b i t o r i s reversed a t low pH, presumably as a r e s u l t o f α-amylase d e s t r u c t i o n as i n d i c a t e d above. I n v e s t i g a t i o n o f the s t o i c h i o m e t r y o f i n t e r a c t i o n has shown that hog-pancreatic α-amylase and i n h i b i t o r combine in a 1:1 f a s h i o n . Complex formation has been demonstrated d i r e c t l y by molecular-sieve chromatography on Sephadex G-100 a f t e r pre-incubation o f s t o i c h i o m e t r i c amounts o f α-amylase and i n h i b i t o r ( F i g . 2). Phaseolus vulgaris α-amylase i n h i b i t o r has a molecular weight o f approximately 45,000, t h i s being somewhat lower than the value reported by Jaffé and co-workers (52). At the present time nothing i s known about the r o l e o f f u n c t i o n a l group(s) c o n t r i b u t i n g t o the a c t i v i t y o f the i n h i b i t o r . Physiological Inhibitors

Effects

and Nutritional

Significance

of v.-Amylase

The presence o f high l e v e l s o f α-amylase i n h i b i t o r s i n common plant f o o d s t u f f s , p a r t i c u l a r l y legumes and c e r e a l s , r a i s e s the question whether these i n h i b i t o r s can have any e f f e c t on aamylases in vivo and, i f so, whether t h i s r e s u l t s i n impaired starch digestion. M i l l e r and Kneen (27) suggested that the polyphenolic i n h i b i t o r i n sorghum i s u n l i k e l y t o have an e f f e c t in vivo, because i t i s r a t h e r r e a d i l y rendered i n e f f e c t i v e as a r e s u l t o f i t s a b i l i t y t o combine w i t h many p r o t e i n s besides α-amylase. However, brown sorghums, which contain high l e v e l s o f t a n n i n s , have been shown t o r e t a r d growth o f c h i c k s , and t o i n t e r f e r e w i t h dry matter d i g e s t i b i l i t y i n r a t s , more than does white (low tannin) sorghum (62-66). The e f f e c t on growth, and f o o d s t u f f d i g e s t i b i l i t y , may be caused by the t a n n i n s , and might w e l l e x p l a i n the b i r d - r e s i s t a n t p r o p e r t i e s o f brown sorghums (62). I t might a l s o be noted that Mandels and Reese (15) b e l i e v e that t a n n i n - c o n t a i n i n g p l a n t s a r e l i k e l y t o be undesirable foods f o r ruminants because o f the i n h i b i t i o n o f rumen c e l l u l a s e by tannins (17). Grazing animals, and even s n a i l s , avoid p l a n t s high i n t a n n i n s , perhaps because o f t h e i r i n h i b i t o r y e f f e c t on h y d r o l y t i c enzymes (67). The s u s c e p t i b i l i t y t o p r o t e o l y s i s o f the proteinaceous aamylase i n h i b i t o r from wheat has been mentioned above. On the b a s i s o f t h i s f i n d i n g , Kneen and Sandstedt (37) concluded that i t would be i n a c t i v a t e d by pepsin i n the stomach, and that the only


260

PHYSIOLOGICAL

< Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 4, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/bk-1975-0015.ch016

7L

EFFECTS

O F FOOD

CARBOHYDRATES

1.0

0.5 ι

0.8 -

0.4

0.6

0.3

0.4

Ο 0.2 >-

Ο &f 0.2 -Jo

_J_

10

20

30

40

50

60

70

80 90

FRACTION NUMBER Figure 1. Purification of Phaseolus vulgaris α-amylase inhibitor by chromatogra phy on CM-cellulose (23)

15

10 v

o

20

25

30

35

FRACTION NUMBER

Figure 2. Molecular sieve chromatography on Sephadex G-100 of hog pancreatic α-amylase (Δ), Phaseolus vulgaris a-amylase inhibitor (•), and a-amylase / inhibitor complex ( O ) ( ) 23



16.

MARSHALL

α- Amyhse Inhibitors from Phnts

261

e f f e c t t h e i n h i b i t o r w o u l d have in vivo w o u l d be on s a l i v a r y a a m y l a s e . They t h e r e f o r e s u g g e s t e d t h a t t h e wheat I n h i b i t o r w o u l d have l i t t l e o r no s i g n i f i c a n c e i n normal human p h y s i o l o g y , and t h a t o n l y i n cases o f impaired p r o t e o l y s i s would i n t e r f e r e n c e w i t h s t a r c h h y d r o l y s i s be a n t i c i p a t e d . S h a i n k i n and B i r k (42) came t o t h e same c o n c l u s i o n . E v i d e n c e I s , however, now a c c u m u l a t i n g t o s u g g e s t t h a t t h e p r o t e i n a c e o u s i n h i b i t o r s o f α-amylase may p l a y an i m p o r t a n t r o l e i n m o d u l a t i n g s t a r c h d i g e s t i o n in vivo. I t h a s been s u g g e s t e d t h a t wheat a m y l a s e I n h i b i t o r s may be, a t l e a s t i n p a r t , r e s p o n s i b l e f o r the d i f f e r e n c e s In n u t r i t i o n a l v a l u e o f d i f f e r e n t v a r i e t i e s o f w h e a t , a s measured by t h e r e t a r d a t i o n o f d e v e l o p m e n t o f Tribolium oastaneum ( H e r b s t ) l a r v a e , compared w i t h t h e o p t i m a l development a c h i e v e d on a s t a n d a r d minimal a r t i f i c i a l d i e t ( 6 8 ) . Recent work has shown In a more c o n v i n c i n g f a s h i o n t h a t t h e a a m y l a s e I n h i b i t o r I n wheat w i l l I n t e r f e r e w i t h s t a r c h m e t a b o l i s m in vivo. In t h i s L a b o r a t o r y I t h a s been shown t h a t c e r t a i n human and a n i m a l f o o d s t u f f s c o n t a i n h i g h l e v e l s o f α-amylase I n h i b i t o r a c t i v i t y (69). R a t s f e d on a d i e t o f o n e l a b o r a t o r y a n i m a l f o o d s t u f f ( R a l s t o n P u r i n a r a t chow) I n w h i c h t h e i n h i b i t o r had been I n a c t i v a t e d by a u t o c l a v i n g , grew a t a r a t e 15-20% f a s t e r t h a n d i d r a t s f e d on t h e u n t r e a t e d f e e d . S i m i l a r l y , when g r o u p s o f r a t s w e r e f e d on a c a s e i n / s t a r c h d i e t i n t h e p r e s e n c e and a b s e n c e o f p u r i f i e d wheat I n h i b i t o r , r e d u c t i o n o f g r o w t h r a t e and I n c r e a s e d f e c a l s t a r c h l e v e l s w e r e c a u s e d by t h e p r e s e n c e o f i n h i b i t o r , t h e m a g n i t u d e o f t h e s e changes b e i n g d e p e n d e n t o n t h e i n h i b i t o r d o s a g e ( 7 0 ) . Added I n h i b i t o r d i d n o t have a n y e f f e c t when s u c r o s e r e p l a c e d s t a r c h a s d i e t a r y c a r b o h y d r a t e ; neither did Inactivated Inhibitor a f f e c t starch u t i l i z a t i o n . Strumeyer (45) has r e c e n t l y s u g g e s t e d t h a t a m y l a s e I n h i b i t o r s may be r e s p o n s i b l e f o r t h e s e n s i t i v i t y t o wheat a n d r y e f l o u r In c e l i a c disease - a gluten-induced enteropathy. Individuals with this d i s o r d e r e x h i b i t i m p a i r e d a b i l i t y t o m e t a b o l i z e s t a r c h and a b s o r b fats. These n u t r i e n t s a r e e x c r e t e d and t h e p a t i e n t s s u f f e r f r o m v i t a m i n d e f i c i e n c y and m a l n u t r i t i o n . A c c o r d i n g t o S t r u m e y e r (45) t h e c e r e a l a m y l a s e i n h i b i t o r s m i g h t be r e s p o n s i b l e f o r t h e s e n s i t i v i t y t o wheat and r y e f l o u r s by d e p r e s s i o n o f an a l r e a d y d e f i c i e n t p a n c r e a t i c α-amylase s u p p l y . I t i s p r o b a b l e t h a t bean α-amylase i n h i b i t o r i s a l s o a c t i v e in vivo. V e n e z u e l a n w o r k e r s have d e t e c t e d l a r g e amounts o f u n d i g e s t e d s t a r c h , and a m y l a s e i n h i b i t o r y a c t i v i t y , i n t h e f e c e s o f r a t s f e d on d i e t s o f raw k i d n e y beans ( 6 1 , 7 1 ) . Evans and c o w o r k e r s have s t u d i e d t h e e f f e c t o f d i f f e r e n t p r o t e i n f r a c t i o n s f r o m Phaseolus vulgaris on t h e g r o w t h o f r a t s . They c o n c l u d e d t h a t t h e r e I s p r e s e n t i n beans a g r o w t h i n h i b i t o r y f a c t o r d i s t i n c t from the o t h e r t o x i c f a c t o r s , phytohemagglutInins and p r o t e a s e I n h i b i t o r s ( 7 2 , 7 3 ) . Many o f t h e p r o p e r t i e s o f t h i s g r o w t h i n h i b i t o r y p r o t e i n (73) r e s e m b l e t h o s e o f t h e Phaseolus vulgaris α-amylase i n h i b i t o r p u r i f i e d and c h a r a c t e r i z e d I n t h i s Laboratory (23).



PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES

Δ BG (mg/100 ml) +40

+20

h or—

-20 Δ IRI (juU/ml) +40 h

•*

/A

+20

L

0

15 30 45 60

90

180

120 minutes

Diabetologia

Figure 3. Elevation of blood glucose (ABG) and serum insulin (MRI) levels in healthy human subjects following intake of starch (100 g) in the absence of wheat a-amylase inhibitor (*) and in the presence of inhibitor (φ, 350 mg; A, 700 mg). Results shown are the mean for seven subjects (75).



16.

MARSHALL

a-Amyhse Inhibitors from Phnts

263

In view o f t h e i r in vivo a c t i o n , the presence o f α-amylase i n h i b i t o r s in f o o d s t u f f s prepared from p l a n t s such as wheat, rye and bean&, i s c l e a r l y undesirable when maximum n u t r i t i o n a l value of the f o o d s t u f f i s required. Wheat amylase i n h i b i t o r has been reported t o p e r s i s t through bread baking, being found i n large amounts in the centers o f loaves (37, 7*0. In t h i s Laboratory i t has been shown that a number o f common l a b o r a t o r y animal feeds, of which wheat i s a major c o n s t i t u e n t , contain i n h i b i t o r a c t i v i t y (69). The same i s true o f a number o f wheat-based human breakfast c e r e a l s (69). It would seem l i k e l y that the true c a l o r i c value o f these f o o d s t u f f s w i l l be lower than expected on the basis o f t h e i r s t a r c h contents, because o f the i n t e r f e r e n c e with s t a r c h d i g e s t i o n caused by endogeneous i n h i b i t o r . The r e l a t i v e l y s t a b l e nature o f α-amylase i n h i b i t o r s means that s p e c i a l a t t e n t i o n w i l l be required to ensure t h e i r d e s t r u c t i o n o r removal during food processing. F i n a l l y , i t i s appropriate t o consider α-amylase i n h i b i t o r s as p o s s i b l e t h e r a p e u t i c agents. The a b i l i t y o f these substances to block s t a r c h degradation by α-amylase in vivo suggests t h e i r p o s s i b l e use as novel d i e t e t i c agents, o f use in the therapy o f o b e s i t y in subjects who f i n d d i f f i c u l t y in completely e l i m i n a t i n g s t a r c h from t h e i r d i e t s . A second a p p l i c a t i o n would be t o a l l o w d i a b e t i c i n d i v i d u a l s t o consume a moderate amount o f s t a r c h , i f ingested in the presence o f i n h i b i t o r . The e f f e c t o f the i n h i b i t or on s t a r c h d i g e s t i o n w i l l help prevent the elevated blood sugar l e v e l which normally r e s u l t s from s t a r c h i n t a k e , and which i s undesirable in the d i a b e t i c . Puis and h i s co-workers {kh) have patented a wheat α-amylase I n h i b i t o r p r e p a r a t i o n , and have proposed i t s use as an antihyperglycemlc agent. Their s t u d i e s w i t h t h i s I n h i b i t o r provide the most convincing evidence o f the in vivo e f f e c t o f an α-amylase i n h i b i t o r , and support i t s p o s s i b l e use as a t h e r a p e u t i c agent. On a d m i n i s t r a t i o n o f the wheat i n h i b i t o r t o r a t s , dogs and healthy human s u b j e c t s , the hyperglycemia and hyperînsulinemla r e s u l t i n g from s t a r c h loading could be reduced dose dependently by the i n h i b i t o r (75). The e f f e c t o f i n h i b i t o r on the l e v e l s o f blood glucose and i n s u l i n in human subjects f o l l o w i n g i n g e s t i o n of raw s t a r c h Is shown in F i g . 3. When cooked, as opposed t o raw, s t a r c h was used, the smoothing e f f e c t on hyperglycemia was somewhat reduced. The suggestion was made that t h i s might be because the a f f i n i t y o f amylase f o r g e l a t i n i z e d s t a r c h i s greater than the a f f i n i t y f o r the i n h i b i t o r , and i t was suggested that an i n h i b i t o r w i t h g r e a t e r a f f i n i t y f o r α-amylase might be advantageous f o r t h e r a p e u t i c use. Acknowledgements Helpful d i s c u s s i o n with Dr. W.J. Whelan i s g r a t e f u l l y acknow ledged. J.J. Marshall i s an I n v e s t i g a t o r o f Howard Hughes Medical Insti tute.


264

PHYSIOLOGICAL EFFECTS OF FOOD CARBOHYDRATES


Literature Cited

1. FRITZ, H. and TSCHESCHE, H. (eds.), Proceedings of the International Conference on Proteinase Inhibitors, Munich, Nov. 1970, Walter de Gruyter, Berlin, 1971. 2. WERLE, E. and ΖICKGRAF-RÜDEL, G., Z. Klin. Chem. u. Klin. Biochem. (1972) 4, 139-150. 3. RYΑΝ, C.A., Ann. Rev. Plant Physiol. (1973) 24, 173-196. 4. RÜHLMANN, Α., KUKLA, D., SCHWAGER, P., BARTELS, K. and HUBER, R., J. Mol. Biol. (1973) 77, 417-436. 5. SCHWIMMER, S., MAKAVER, R.U. and ROREM, E.S., Plant Physiol. (1961) 36, 313-316. 6. PRESSEY, R., Arch. Biochem. Biophys. (1966) 113, 667-674. 7. PRESSEY, R., Plant Physiol. (1967) 42, 1780-1786. 8. PRESSEY, R. and SHAW, R., Plant Physiol. (1966) 4l, 1657-1661. 9. JAYNES, T.A. and NELSON, O.E., Plant Physiol. (1971) 47, 629-634. 10. WINKENBACH, F. and MATILE, P., Z. Pflanzenphysiologie (1970) 63, 292-295. 11. ALBERSHEIM, P. and ANDERSON, A.J., Proc. Nat. Acad. Sci. U.S. (1971) 68, 1815-1819. 12. ANDERSON, A.J. and ALBERSHEIM, P., Physiol Plant Pathol. (1972) 2, 339-346. 13. FISHER, M.L., ANDERSON, A.J. and ALBERSHEIM, P., Plant Physiol. (1973) 51, 489-491. 14. MANDELS, M., HOWLETT, W. and REESE, E.T., Can.J.Microbiol. (1961) 7, 957-959. 15. MANDELS, M. and REESE, E.T., in REESE, E.T. (ed.), Advances in Enzymic Hydrolysis of Cellulose and Related Materials, pp.115-157,Pergamon Press, Oxford, 1963. 16. MANDELS, M. and REESE, E.T., Ann. Rev. Phytopathol. (1965) 3, 85-102. 17. SMART, W.W.G., BELL, T.A., STANLEY, N.W. and COPE, W.A., J. Dairy Sci. (1961) 44, 1945-1946. 18. KING, K.W., Va. Agric. Exp. Sta. Tech. Bull. 127, December 1956. 19. BERNHEIMER, A.W. and STEELE,J.M.,Proc. Soc. Exp. Biol. Med. (1955) 89, 123-126. 20. BERNFELD, P., in COLOWICK, S.P. and KAPLAN, N.O. (eds.), Methods in Enzymology, Vol. 1, pp. 149-158, Academic Press, New York, 1955. 21. NELSON, Ν., J. Biol. Chem. (1944) 153, 375-380. 22. HOPKINS, R.H. and BIRD, R., Biochem.J.(1954) 56, 86-99. 23. LAUDA, C.M. and MARSHALL, J.J., in preparation (1975). 24. F0SSUM, K. and WHITAKER, J.R., J. Nutr. (1974) 104, 930-936. 25. HEMBERG, T. and LARSS0N, I., Physiol. Plant(l961)14, 861-867. In Physiological Effects of Food Carbohydrates; Jeanes, Allene, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

16.

MARSHALL

α-Amylase

Inhibitors

from

Plants

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