Physiological Effects of Food Carbohydrates - ACS Publications

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14 The Lysosomal α-Glucosidases of Mammalian Tissues Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 10, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/bk-1975-0015.ch014

BARBARA I. BROWN, A L L E N K. MURRAY, and DAVID H. BROWN Department of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University, St. Louis, Mo. 63110

This Symposium has focused attention on various dietary car­ bohydrates, their absorption, interconversions and physiological fates. As has been pointed out, one of the criteria for utiliza­ tion of [ C]-labeled sugars has been the extent of isotope in­ corporation into glycogen as well as the formation of CO . The average 70 kg adult may have stored in his tissues half a kilogram of glycogen. However, a 7 kg infant with Type II Glycogen Storage Disease (Pompe's disease), which is one of the more common forms of glycogen storage disease, also may have stored a like quantity of glycogen. This fact serves to emphasize the importance of the α-glucosidase which has an acidic pH optimum in the catabolism of glycogen in normal human tissues, since patients with Type II disease have been shown to have a congenital and generalized de­ ficiency of this glucosidase (1). The purified enzyme has both α-1,4 and α-1,6 glucosidase activity and can convert glycogen totally to glucose (2). The activity in normal tissue homogenates is such that if the enzyme were in contact with its substrate, it would have the capacity of totally degrading the polysaccharide present in liver in 3 to 5 hours and that in muscle in 8 to 12 hours. 14

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By comparison o f i t s p r o p e r t i e s w i t h those o f an a - g l u c o s i dase p u r i f i e d t o homogeneity from r a t l i v e r lysosomes ( 3 ) , the human enzyme i s assumed t o be i n lysosomes a l s o . E l e c t r o n micro­ graphs o f l i v e r samples obtained from p a t i e n t s w i t h Type I I g l y ­ cogen storage disease show t h a t a l a r g e q u a n t i t y o f glycogen i s present w i t h i n membrane enclosed vacuoles and t h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h the hypothesis t h a t the α-glucosidase has a l y s o ­ somal l o c a l i z a t i o n ( 4 ) . S i m i l a r enzymes have been p u r i f i e d from a v a r i e t y of sources such as r a t C2,_5,6) and beef l i v e r ( 7 ) , rab­ b i t muscle ( 8 ) , and human p l a c e n t a (6) and l i v e r . Most of the p r e p a r a t i o n s have taken advantage o f the property o f the enzyme to be s p e c i f i c a l l y r e t a r d e d by a d s o r p t i o n to Sephadex as an im­ p o r t a n t a i d i n i t s p u r i f i c a t i o n . F i g u r e 1 i l l u s t r a t e s the behav­ i o r o f the human l i v e r enzyme upon Sephadex G-100 chromatography. Previous steps i n the p u r i f i c a t i o n procedure had i n c l u d e d repeated 223 Jeanes and Hodge; Physiological Effects of Food Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

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f r e e z i n g and thawing of the l i v e r homogenate to rupture the l y s o ­ somes, a heat treatment at 55° and ammonium s u l f a t e f r a c t i o n a t i o n between 35 and 55% s a t u r a t i o n . The d i a l y z e d , concentrated ammo­ nium s u l f a t e f r a c t i o n a p p l i e d to the Sephadex column u s u a l l y con­ t a i n e d 60% of the s t a r t i n g u n i t s of enzyme a c t i v i t y and had a s p e c i f i c a c t i v i t y of 0.15 u n i t per mg p r o t e i n corresponding to i t s being 10 to 1 2 - f o l d p u r i f i e d at t h i s stage. However, the en­ zyme recovered from the Sephadex column u s u a l l y had an a c t i v i t y of about 30 u n i t s per mg corresponding to an o v e r a l l p u r i f i c a t i o n of more than 2000-fold w i t h a recovery of approximately 40% of the t o t a l u n i t s . From 1 to 2 mg of enzyme could be obtained from 100 grams of human l i v e r ( 9 ) . As i n d i c a t e d i n F i g u r e 1, maltose or glycogen can be used as a s u b s t r a t e i n measuring the a c t i v i t y of the enzyme. The s p e c i f ­ i c a c t i v i t i e s given above r e f e r to ymoles of maltose hydrolyzed per minute per mg of p r o t e i n at pH 4 and 37° and at a s u b s t r a t e c o n c e n t r a t i o n of 10 mM. U s u a l l y incubations w i t h glycogen were c a r r i e d out w i t h 1% s u b s t r a t e , at pH 4.5, and the a c t i v i t i e s then expressed as ymoles of glucose formed per minute at 37°. The p l a ­ c e n t a l enzyme p u r i f i e d by de Barsy et a l . (6) had a s p e c i f i c ac­ t i v i t y of 7.3 u n i t s per mg when assayed at 3.7 mM maltose. How­ ever, these i n v e s t i g a t o r s found that the 1^ f o r the human p l a c e n ­ t a l enzyme was 11 mM f o r maltose and 2% f o r glycogen. Even though glycogen i s the n a t u r a l s u b s t r a t e f o r the en­ zyme, f o r convenience many i n v e s t i g a t o r s have used maltose as a s u b s t r a t e i n assaying f o r the α-glucosidase. We had p r e v i o u s l y shown t h a t the r a t l i v e r enzyme e x h i b i t e d s u b s t r a t e i n h i b i t i o n by maltose and by m a l t o s i d i c a l l y l i n k e d o l i g o s a c c h a r i d e s when the i n i t i a l s u b s t r a t e concentrations exceed 5 to 10 mM ( 2 ) . Studies w i t h the enzyme p u r i f i e d from human l i v e r have revealed no sub­ s t r a t e i n h i b i t i o n a t concentrations as h i g h as 100 mM maltose. The Kflj f o r maltose f o r the human l i v e r enzyme appears to be 9 mM, w h i l e f o r isomaltose a Km of 33 mM was found. Polyacrylamide g e l e l e c t r o p h o r e s i s of the p u r i f i e d human l i v e r enzyme r e s u l t e d i n a s i n g l e peak of a c t i v i t y as shown i n F i g u r e 2. In t h i s experiment a f t e r e l e c t r o p h o r e s i s the g e l was f r o z e n , s l i c e d i n t o 1 mm s l i c e s and the a c t i v i t y of the e l u t e d en­ zyme determined i n each s l i c e . E s s e n t i a l l y 100% of the loaded u n i t s were recovered. Of g r e a t e r i n t e r e s t i s the f a c t t h a t a good correspondence was found between t o t a l absorbance at 280 nm, pro­ t e i n s t a i n i n g w i t h Coomassie b l u e , enzymatic a c t i v i t y revealed by i n c u b a t i o n w i t h methyl u m b e l l i f e r y l glucopyranoside (another sub­ s t r a t e of the α-glucosidase), and PAS p o s i t i v e m a t e r i a l i n the same or p a r a l l e l g e l s . Since PAS s t a i n i n g i n d i c a t e s the presence of carbohydrate, the α-glucosidase i s o l a t e d from human l i v e r ap­ pears to be a g l y c o p r o t e i n ( 9 ) . In i s o e l e c t r i c f o c u s i n g g e l s there always was evidence of the presence of s e v e r a l isozymes as i n d i c a t e d i n F i g u r e 3 where the e l u t e d enzyme showed s i m i l a r p a t t e r n s of a c t i v i t y w i t h the two s u b s t r a t e s . The pH values were obtained by measuring the pH of

Jeanes and Hodge; Physiological Effects of Food Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

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water i n which groups of four consecutive s l i c e s of a d u p l i c a t e g e l were e x t r a c t e d f o l l o w i n g overnight e l e c t r o p h o r e s i s o f a 17 cm g e l c a s t w i t h pH 3 t o 6 ampholine. D u p l i c a t e g e l s were a l s o s t a i n e d f o r p r o t e i n and PAS r e a c t i v e m a t e r i a l . The scans of t h e s t a i n e d g e l s (Figure 4) i n d i c a t e d correspondence between p r o t e i n and carbohydrate throughout the r e g i o n c o n t a i n i n g the charge i s o ­ zymes. F o l l o w i n g column chromatography o f the glucosidase on B i o g e l P-200, some d i f f e r e n c e i n the isozyme p a t t e r n o f i n d i v i d u a l f r a c t i o n s could be a s c e r t a i n e d i n i s o e l e c t r i c focused g e l s such t h a t the more a c i d i c isozymes were more prominent i n the e a r l i e r f r a c t i o n s e l u t e d from the column w h i l e the l e s s a c i d i c ones emerged l a t e r . The p o s i t i o n o f the main peak i s compatible w i t h a molecular weight on the order of 100,000 s i m i l a r t o the v a l u e of 114,000 found f o r the r a t l i v e r lysosomal α-glucosidase (3) and of 107,000 found f o r the bovine l i v e r enzyme ( 7 ) . One o f the main purposes o f p u r i f y i n g the human l i v e r enzyme was t o make i t p o s s i b l e t o o b t a i n an antibody t o the p r o t e i n t o i n v e s t i g a t e whether, u s i n g such an antibody, evidence could be found f o r c r o s s - r e a c t i v e m a t e r i a l i n the t i s s u e s o f i n d i v i d u a l s a f f l i c t e d w i t h Type I I glycogen storage d i s e a s e . The evidence t h a t the α-glucosidase has an e s s e n t i a l r o l e i n normal metabolism has been i n f e r r e d from the apparent s e r i o u s e f f e c t s o f i t s ab­ sence i n the t i s s u e s o f the i n f a n t w i t h t h i s disease who f r e ­ quently d i e s a t an e a r l y age w i t h massive accumulation o f g l y ­ cogen p a r t i c u l a r l y i n the heart and s k e l e t a l muscles. However i t should be pointed out t h a t a d u l t forms o f the d e f i c i e n c y a l s o e x i s t (10) and, w h i l e these a d u l t s have e s s e n t i a l l y no demonstra­ b l e α-glucosidase a c t i v i t y a t a c i d pH i n e x t r a c t s o f t h e i r t i s ­ sues, the content o f glycogen may be o n l y s l i g h t l y above normal and the c l i n i c a l symptoms may be confined t o a m i l d muscular weak­ ness. Since the a c t i v i t y of the α-glucosidase i s demonstrable i n leukocytes and c u l t u r e d s k i n f i b r o b l a s t s o f normal i n d i v i d u a l s , f i b r o b l a s t s may be u t i l i z e d as an experimental t i s s u e (11,12). Rabbits were immunized by subcutaneous i n j e c t i o n of 0.5 t o 1 mg o f enzyme i n 1 ml o f complete Freunds adjuvant i n m u l t i p l e s i t e s a t monthly i n t e r v a l s f o l l o w e d by 0.1 mg of enzyme i n t r a ­ venously. The t i t e r o f antibody obtained v a r i e d w i t h the r a b b i t and the time o f b l e e d i n g d u r i n g the immunization schedule. A t y p i c a l p r e p a r a t i o n o f antibody was 5 t o 10 times as e f f e c t i v e i n i n h i b i t i n g the a c t i v i t y of the enzyme toward glycogen as toward e i t h e r maltose o r isomaltose when a l l a c t i v i t i e s were assayed a t c o n c e n t r a t i o n s equal t o the Km o f the r e s p e c t i v e s u b s t r a t e s . Ac­ t i o n on glycogen could be completely i n h i b i t e d by antibody, w h i l e i n general no more than 80% o f maltose h y d r o l y s i s could be blocked even by l a r g e amounts o f antibody. S i m i l a r e f f e c t s were observed by deBarsy e t a l . (6). In g i v i n g a t t e n t i o n next t o some of the experimental r e s u l t s obtained u t i l i z i n g f i b r o b l a s t s , i t i s s i g n i f i c a n t t o recognize t h a t u n i t s o f a c t i v i t y are expressed as nanomoles o f maltose h y d r o l y z e d , o r glucose formed from glycogen, per minute per mg of

Jeanes and Hodge; Physiological Effects of Food Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

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G-100 Figure 1. Chromatography of human liver a-glucosidase on Sephadex G-100. See text for assay conditions.

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