Advances in Enzymology and Related Areas of Molecular Biology, Volume 63 Edited by Alton Meister Copyright © 1963 by John Wiley & Sons, Inc.

INSULIN AND THE STIMULATION OF GLYCOGEN SYNTHESIS. THE ROAD FROM GLYCOGEN STRUCTURE TO GLYCOGEN SYNTHASE TO CYCLIC AMP-DEPENDENT PROTEIN KINASE TO INSULIN MEDIATORS By JOSEPH LARNER*, Department of Pharmacology, Jordan Hall, University of Virginia School of Medicine, Charlottesville, Virginia 22908 CONTENTS I.

11.

111.

IV .

V.

VI. VII. VIII.

IX.

Introduction Historical A. Discovery B. Insulin and Glycogen and Fat Synthesis C. Glycogen Storage Diseases D. Chemical and Polymeric Structure E. Enzymology F. Glycogen in the Cell Biochemical Considerations and a Glycogen-K+ Pump Hypothesis Integration of Covalent and Allosteric Controls of Phosphorylase and Glycogen Synthase Phosphorylation Sites of Glycogen Synthase Emerging Significance of Multiple Phosphorylation Decreased Phosphorylation of Glycogen Synthase with Insulin Action Insulin and Established Second Messengers A. Cyclic AMP (CAMP) B. Cyclic GMP (cGMP) C. Cat' and Phosphoinositides Stable and Allosteric Effects of Insulin on Protein Kinases A. General B. Insulin and Insulin Receptor Tyrosine Kinase

This work was supported in part by NIH Grant 5-R37-DK14334-20 and Pratt Fund University of Virginia. This Chapter is dedicated to the many co-workers who participated in the research and to the colleagues whose suggestionsand criticisms have been so much appreciated over the years.

173

174

X. XI. XII. XI11. XlV. XV.

JOSEPH LARNER

C. Insulin and MAP Kinase D. Insulin and Casein Kinases 1 and 2 E. Insulin and Protein Kinase C F. Insulin and CAMP-Dependent Protein Kinase-A Marker for Mediator Short- and Long-Term Effects of Insulin on Phosphoprotein Phosphatases Two Pathways for Activation of Glycogen Synthase by Insulin Purification and Action of Two Mediators-A Mechanism for Dephosphory lation Formation of lnsulin Mediators Insulin Receptor Activation and Mediator Formation Summary References

I. Introduction It can be argued from the outset that glycogen has been a major catalyst for formulation of many concepts of modem biology. Glycogen was the first biopolymer synthesized in a test tube (so-called “blue” glycogen because it stained blue with iodine), and the first biopolymer whose structure was determined by specific enzymatic degradation methods. Glycogen storage was the first inborn error of metabolism to be directly shown to be due to a defect in an enzyme. Studies of the control of glycogen synthesis and degradation have been intimately related to investigations of the mechanisms of insulin and glucagon action. A number of broad principles have emerged from studies of glycogen metabolism. First, covalent phosphorylation was originally discovered as the mechanism of activation for phosphorylase. Second, phosphorylase b kinase was discovered to be analogously controlled by phosphorylation. The third enzyme, glycogen synthase, was then found to be inversely controlled by phosphorylation, that is, inactivated. When mitochondrial pyruvate dehydrogenase was next discovered to be controlled in a similar manner to glycogen synthase, it became apparent that phosphorylation control spread beyond the bounds of glycogen metabolism where it was believed to be confined. The discovery of cyclic AMP (CAMP)and the development of the concept of second messengers came from studies on the control of phosphorylase by glucagon and epinephrine, which also led to the concept of the cascade of phosphorylation reactions. Finally, the concept and emerging significance of multiple phos-

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phorylation occurred via studies on glycogen synthase. Further studies of the dephosphorylation of glycogen synthase by insulin as related to the inhibition of the CAMP-dependent protein kinase led to the discovery of novel inositol glycan insulin mediator molecules. Thus glycogen has sparked its fair share of major discoveries in biology. In this chapter we will focus on the mechanisms of glycogen metabolism controlled by insulin. 11. Historical A. DISCOVERY

Glycogen was discovered and named by C. Bernard in 1870, who noted its ability to provide blood glucose (1). The name “matiere glycogene” meant glucose forming material. It was present in liver as a water insoluble material. After standing for some time, glycogen was converted in the liver to water soluble glucose. Bernard observed this by perfusing the liver with water. When he prepared a “filtered decoction” of liver (hot water extract) he frequently noted a marked opal color. When he added alcohol to the decoction a white precipitate formed that was collected and dried. Analysis showed it to be similar to starch. Its properties, as described by Bernard, are given in Table 1 and were found to be essentially correct and complete. When chemically analyzed by Pelouze for its elements it was found to be similar to starch (Table 2), but there were some obvious discrepancies apparent. B. INSULIN AND GLYCOGEN AND FAT SYNTHESIS

In 1921, Banting and Best succeeded in identifying and isolating insulin by its ability to lower blood glucose in diabetic dogs (2). One TABLE I Properties of Glycogen (C. Bernard) Color whitish, opaline, milky Iodine color red wine color, between starch blue and dextrin red Precipitated with alcohol Nonreducing to alkaline cupric potassium Hydrolyzed by mineral acids, superheated steam Sensitive to salivary diastase and diastase vegetale (Payen and Persoz 1840) When hydrolyzed it was converted to sugar, and became reducing

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TABLE 2 Analysis of Sugars, Starch, and Glycogen (Pelouze) Cellulose C1ZH1OOIO Starch C 12H10010 Dextrine C12H10010 + aq Glycogene C12H10010 + 2H0 Cane Sugar ClZH11011 Glucose C12H12012

year later Banting et al. (3) noted that insulin administration in vivo increased glycogen and fat stores in liver, fat, and other tissues, demonstrating that insulin promoted glucose utilization by the tissues for synthesis of these products. The respiratory quotient was simultaneously increased indicating that energy had been produced to drive these reactions. C. GLYCOGEN STORAGE DISEASES

In 1928 van Creveld reported a syndrome in an 8-year old boy who had an enlarged liver, hypoglycemia, and ketonuria (4). He reasoned that the breakdown of glycogen to blood sugar was impaired since his blood sugar failed to rise except minimally after the administration of adrenalin. Glycogen synthesis was thought to be normal, since after the administration of glucose, the respiratory quotient rose to about unity. From the work of Wilder, it was known at that time that hyperinsulinism could be associated with storage of glycogen and a poor response.to adrenalin. van Creveld was then able to show that his patient did not have altered sensitivity to insulin. From these experiments he concluded that the patient probably was unable to degrade glycogen to sugar, but was able to carry out the synthesis of glycogen from sugar. In 1929 von Gierke published the clinical and pathological findings in the cases of two children with abnormally large deposits of glycogen in their livers and kidneys (5). Chemical studies performed by Schoenheimer, who was later to become well known for his pioneering work with isotopes in metabolism, characterized the glycogen chemically (6). In addition, he did the now classic experiment of simply incubating liver homogenates to decide whether the glycogen itself or the enzymes were abnormal in this disease. When he

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incubated the control liver homogenate, the glycogen present was broken down by the enzymes. When he incubated the liver homogenate from the patient’s liver he found that the glycogen did not break down. When he mixed the two liver samples, the glycogen of the patient’s liver was broken down by the enzymes of the normal liver. He concluded that the liver enzymes rather than the glycogen itself were responsible for the disease. This experiment was inherently simple and incisive. It is most likely that the enzyme defect that Schoenheimer studied was a deficiency of debranching enzyme amylo-1,4 -+ 1,4-transglycosylase/ amylo-1,6-glucosidase rather than glucose-6-phosphatase (G-6P) as was previously thought. Thus, a defect of G-6-P would not lead to an impaired enzymatic breakdown of glycogen in a broken cell system since this enzyme is not itself directly involved in the breakdown of glycogen. Gerty Con and co-worker pioneered in establishing the enzymic basis(es) for glycogen storage diseases (7); this was the last and possibly the most important work of the woman who began her career in pediatrics. In 1952 she demonstrated that the livers and kidneys of patients similar to the ones described by von Gierke and van Creveld were deficient in the enzyme G-6-P. As was stated in a eulogy by Ochoa and Kalckar (8); “Gerty Cori’s studies on the glycogen storage diseases represent the first demonstration that a human hereditable disease stems from a defect in an enzyme.” D. CHEMICAL AND POLYMERIC STRUCTURE

Since Bernard, the chemistry of glucose itself and of the interglucose linkages of starch and glycogen have been elucidated. Starch fractions and glycogen were studied by methylation techniques. The methylated polysaccharides were hydrolyzed with acid to the constituent methylated sugars. Wherever there were linkages present in the polysaccharide no methyl groups would be introduced and the position of the linkage could be determined. In 1932 Haworth and Percival(9), reported that the major product of hydrolyzed glycogen was 2,3,6trimethyl glucose and the minor produce (-9%) was 2,3,4,6-tetramethyl glucose, thus proving that the majority of glucose residues in glycogen were linked by 1,4 linkages. The nonreducing end residues, which had free hydroxyl groups in the 4 position, gave 2,3,4,6-tetramethyl glucose. From the proportions of

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the two, that is the ratio of 2,3,6-trimethyl glucose to 2,3,4,6-tetramethyl glucose, a linear structure 12 residues in length was proposed. However, physical chemical studies soon made it clear that these polysaccharide structures were much larger. In 1939 Carter and Record (10) showed, by osmotic pressure measurements, that the average degree of polymerization of a sample of methylated glycogen was 3000 to 4000 glucose residues, whereas the same sample had an average chain length of only 18 by the chemical methylation method. This implied that glycogen had a branched structure. The first clues as to the nature of the branch linkages came from further studies of hydrolysates of methylated glycogen and starch. 2,3-Dimethyl glucose from methylated hydrolyzed starch and glycogen was isolated by several workers including Irvine in 1932 (1l), Bell in 1935 (12), and Haworth, Hirst, and Webb in 1938 (13). The amount of dimethyl glucose was equivalent to the tetramethyl glucose. Dimethyl glucose would be expected from subunits united by a 1,6 linkage. The isolation of the 1,6-linked disaccharide isomaltose from hydrolyzed starch and glycogen in 1947 and 1949 by Montgomery et al. (14) and Wolfrom el al. (15) provided the final proof by chemical identification of the branch points. The arrangement of the branch points in the macromolecule was then studied with enzymes, when it became clear that different enzymes selectively degraded the two different linkages of the molecules. Meyer, Hohenemser, and Bernfeld in 1940 (16) were the first to use two enzymes for the structural determination of the branched component of starch, amylopectin. P-Amylase was used to degrade the 1,4 linkages. The enzyme initiated hydrolysis at the nonreducing ends of the outer chains. At the branch points the hydrolysis ceased. A separate enzyme from yeast, a 1,6-glucosidase, was then used to hydrolyze the branched or 1,6linkages. From a study of the products formed at each step in the degradation, a model with a treelike arrangement of the branch points was proposed. Larner et al. in 1952 (17) next used phosphorylase, and the debranching enzyme of muscle and degraded 4 polysaccharides, rabbit liver and muscle glycogens, and corn and wheat amylopectins (Table 3). In the case of all four polysaccharides a treelike or bushlike pattern of branching was deduced from a quantitative estimation of the products that appeared in each step of the degradation. The muscle glycogen

Rabbit liver glycogen Rabbit muscle glYcogen Wheat amylopectin Corn amylopectin

Pol ysaccharide 5.3 5.1 8.2 12.9

15 18 24

10.8 15.6

7.1

Phosphorylase p-amylase

15

Average chain length

Numbers of residues per outer branch hydrolyzed

13 18

9

9

Outer branches

53 66 61

5 6

30

1

17 29

23

23

2

(%)

15

3

Branch point in successive tiers

6

6

Inner branches

Average number of glucose residues

TABLE 3 Length of Outer and Inner Branches and Distribution of Branch Points

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JOSEPH LARNER

model derived from these studies is shown in Fig. 1. Similar conclusions were also arrived at by Whelan and Roberts using a-amylase and the plant debranching enzyme, R enzyme, and degrading in a simultaneous rather than successive manner (18). There is thus general agreement that these biopolymers have a treelike or bushlike arrangement of branch points in which the number of branch points in each tier decreases as the reducing end of the molecule is approached. These were probably the first biopolymers to be selectively degraded by enzymes for the determination of average inner structure. Today we recognize that the giant molecule is built up on a protein, glycogenin, and that the structure analyzed is a segment of the giant molecule (Fig. 2) (19).

Figure 1. Model of a segment of muscle glycogen based on results obtained by a stepwise enzymatic degradation. 0, 0 and 0 glucose residues removed by first, second, and third degradation with phosphorylase, respectively. 0 , glucose residues removed by amylo-1-6-glucosidase. Of five tiers, three were degraded, corresponding to 122 out of 150 glucose residues. Adopted from reference 17 with permission.

INSULIN AND THE STIMULATION OF GLYCOGEN SYNTHESIS

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Figure 2. Schematic model of glycogen particle. A chains are unbranched. B chains are branched.Numbers refer to layers or tiers. Layers 7 and 8 are missing.The central protein now known as glycogenin is as shown. Adopted from reference 19 with permission. E. ENZYMOLOGY

The roots of enzymology are deeply involved in the polysaccharides. It is considered that the work of Kirkhoff in 1811 on starch hydrolysis by amylase initiated enzymology (20). The several classes of amylases as we know them today come from this beginning. However, so far as the modern enzymology of glycogen is concerned, it probably began with the work of Carl and Gerty Cori. In 1936 they isolated glucose-1-phosphate, from a reaction mixture containing

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glycogen, inorganic phosphate Pi, and an aqueous extract of muscle containing phosphorylase and the nucleotide adenylic acid (adenosine-5’-monophosphate, AMP), which activates this enzyme (21). The stage had also been set by the eminent Polish biochemist Parnas who had also discovered the disappearance of inorganic P with glycogen breakdown in muscle extracts and had coined the term phosphorolysis (22). In this reaction, the 1.4-glycosidic linkage was ruptured with the simultaneous incorporation of Pi into organic linkage. It is only fitting that the name Con ester was applied to glucose-lphosphate. The reverse reaction, namely, the synthesis of polysaccharides from glucose-1-phosphate by the action of phosphorylase was announced almost simultaneously in 1939 to 1940 by Cori, Schmidt, and Cori (23), Ostern and Holmes (24), Kiessling (25), and Hanes (26), the last cited in studies on the enzyme from pea seeds. This was an outstanding achievement in biochemistry and for a number of years it was reasonably assumed that the in vivo function of phosphorylase was to catalyze both the synthesis and the breakdown of the 1,4 linkages of glycogen and starch. Of interest, even in the early studies, was the unusual nature of the phosphorylase. In some unexplained manner it was stimulated by adenosine monophosphate (AMP). Originally, it was thought that adenosine-5’-diphosphate(ADP) was the stimulating agent, but Dr. M. Johnson suggested the possibility of a dismutation reaction in which 2 mol of ADP were converted to one each of AMP and adenosine triphosphate (ATP). This suggestion led to the identification of AMP as the actual stimulating agent of phosphorylase and to the unexpected discovery of the description of a new enzyme, myokinase or adenylate kinase as it is known today, which interconverts these nucleotides. Further work led to the crystallization of phosphorylase by A. A. Green and G. T. Cori in 1943 (27). A second form of the same enzyme was detected and crystallized in 1945 (28). Whereas the first form, the active or a form, was only minimally stimulated by AMP (-35%), the second b or inactive form had no activity in the absence of this cofactor. The nature of the two forms was then unknown. A converting enzyme was known, however, which was capable of transforming the a form into the b form. This enzyme, which acted on phosphorylase as substrate, was called PR enzyme, meaning prosthetic group removing (29). This was later renamed “phosphorylase-rupturing” by Cori with his usual brilliant

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aplomb, when it was discovered that in the conversion reaction the molecular weight of phosphorylase was halved. Today it is recognized as phosphoprotein phosphatase. The biochemistry of this interconversion reaction was elucidated by the work of Sutherland et al. with the enzyme from liver (30), and by Krebs and Fischer with the enzyme from muscle (31). in the conversion of the active to the inactive form, Sutherland and coworkers identified Pi as the other reaction product. With this knowledge, Krebs and Fischer, and also Sutherland and co-worker were then able to demonstrate for the first time the reverse reaction, namely, conversion of the dephosphorylated inactive form to the phosphorylated active form (32). For this conversion reaction a kinase and ATP were required. Krebs and Fischer and their co-worker, determined the stoichiometry of the interconversion reactions in the muscle enzyme system as well as the amino acid sequence at the single phosphorylation site. The amino acid sequence of the monomer is now known (33); the enzyme has been cloned (34), and its structure has been determined by X-ray crystallography (35). As mentioned previously, these two interconversion reactions are catalyzed by separate enzymes, a phosphoprotein phosphatase (phosphorylase phosphatase) and an ATP requiring phosphoprotein kinase (phosphorylase b kinase). Of considerable interest was the demonstration by Krebs, Graves, and Fischer and their co-workers that phosphorylase b kinase is subject to control through activation from an inactive to an active form. The phosphorylation of phosphorylase b kinase by ATP, activation in the presence of Ca2', and activation by a proteolytic relation (with trypsin) constitute separate reactions. The first of these is now known to be stimulated by the adenosine-3',5'-cyclophosphate (CAMP) originally discovered by Sutherland and Rall as an intermediate involved in the action of epinephrine and glucagon (36). A series of enzyme reactions that include the formation of CAMPby adenylate cyclase, activation of the AMP-dependent protein kinase by CAMP,phosphorylation and activation of phosphorylase b kinase by the CAMP-dependent protein kinase, and the phosphorylation of phosphorylase b to form phosphorylase a by phosphorylase b kinase function as a cascade in a manner analogous to the cascade of hydrolytic reactions involved in the coagulation of blood. The ion Ca2' appears prominently in both schemes, and there are more than one entry into each

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cascade. One important difference between them is the fact that whereas the coagulation cascade is made up of hydrolytic reactions that are irreversible, the phosphorylase cascade is more finely regulated by phosphate transfer reactions that can reverse the pattern of events, as well as by modulation of substrate controls. The second reaction stimulated by Ca2' is mediated via calmodulin, which by the work of Cohen (37) is known to be present as one of four subunits in phosphorylase b kinase (38, 39). From the work of Leloir and his school it is now clear that a second enzyme UDPG-glycogen synthase exists in cells that catalyze the synthesis of 1,4 linkages in glycogen. In 1957 Leloir and Cardini demonstrated that the liver enzyme catalyzes the direct transfer of glucose from uridine diphosphoglucose (UDPG) to the nonreducing end of the outer chains of glycogen (40). The nucleotide sugar is a derivative of the familiar Con ester, and is a much more effective glucose donor participating in an irreversible reaction in contrast to the glucose-l-phosphate reaction, which is readily reversible (see Section 111). In 1960 Villar-Palasi and Lamer discovered that UDPG glycogen synthase is also subject to control (41, 42). These workers demonstrated that after treatment of muscle with insulin, the enzyme extracted from the muscle was present in an activated state. Conversation of an inactive to an active form by insulin was proposed. The two forms of synthase were first identified and purified by RosellPerez et al. (43) and have been termed D (dependent) and I (independent). Traut also had presented evidence for two forms of the enzyme (44). The nomenclature refers to the fact that the D or dependent form is inactive in the absence of G-6-P, whereas the I or independent form is active without G-6-P. In a reaction with ATP and a separate kinase enzyme stimulated by adenosine 3',5'-cAMP, the I form is converted to the D (45, 46). The reverse reaction, conversion of the D to I form is also catalyzed by a phosphatase (46). The definitive mechanism of the two interconversion reactions by phosphorylation and dephosphorylation was first demonstrated by Friedman and Lamer (46). It is of interest to recalculate the original data of Table 1 (46) in terms of subsequent work. From a mean value of 1.8 mkmol of 32Pincorporated per unit of enzyme converted, assuming a specific activity of 40 unit/mg for the homogeneous enzyme, and a subunit molecular weight of 85,000 (47)

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a value of 6.2 Pkubunit can be calculated. This may be compared with a value of 6 Phbunit by alkali labile P analysis determined later (48). The glycogen synthase interconversion system including the kinase and phosphatase components is under hormonal control being stimulated by insulin and inactivated by epinephrine. Craig and Larner demonstrated that the two hormones compete with each other for the control of this system (49). The similarity of the glycogen synthase and the phosphorylase interconversion enzyme systems is even more striking. Appleman et al. in Leloir’s laboratory showed that synthase I is converted to a D type of enzyme by Ca2’ in the presence of a protein factor now recognized as calmodulin, and by trypsin treatment as well (50). The glycogen synthase and phosphorylase systems are controlled by similar chemical influences in a manner that activates one while inactivating the other. While this picture is generally correct, it is important to point out that there are exceptions. For example, while insulin and epinephrine compete for the control of the synthase system, no competition has been demonstrated in the phosphorylase system in muscle. Nature has apparently built up an extensive apparatus for maintenance of a very finely regulated control through hormones as well as metabolites. Is it not truly fascinating to reconsider Bernard’s discussion of the synthesis and breakdown of glycogen “From the general point of view one could say that these two acts are the inverse of one another?” How prophetic these words now seem when applied to these two enzyme systems. To complete the discussion of glycogen enzymology, the enzymes that form and degrade the other linkage of glycogen the 1,6 linkage or branch linkage will be discussed. As previously mentioned these branch linkages constitute under 10% of the total linkages present, but their importance lies in the fact that they enhance the solubility of the molecule, preventing it from precipitating and acting as a foreign body in the tissues. Such a condition occurs in glycogen storage disease Type IV in which there is brancher deficiency. As mentioned in the structural studies, enzymes that have specificity directed toward the 1,6 linkages have been detected and studied in animal and plant tissues. These act without phosphate, through hydrolysis or through transfer reactions. The action of two such enzymes of animal tissues termed the debranching enzyme, and the

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JOSEPH LARNER

branching enzyme are well established (51, 52). Through the action of phosphorylase the outer chains of glycogen are broken down to a symmetric limit dextrin configuration consisting of about four glucose molecules (see Fig. 3) (53). This is converted to an asymmetric limit dextrin structure by the 1,4 1,4-transferase action of the debrancher (51). The 1,6-linked single glucose residue is thus exposed and hydrolyzed to free glucose by the hydrolytic activity of the debrancher amylo-1,6-glycosidase. The debrancher contains both enzyme activities in a single polypeptide (54). The polysaccharide chain is then free of obstructions and phosphorylase can continue its degradation of the chain until the next obstructive 1,6 link is reached. Thus the molecule can be essentially completely degraded. GLYCOGEN

IQ

Phosphorylase

I t

0-OQ-c-0-0- -MI+ Phosphorylase Limit-Dextrin (Symmetric) Transferase 0

~O-O-O-~*-O-O+ Phosphorylase limit-Dextrin (Asytytetric)

1

Amylo-1, 6-glucosidase

0

+

o-o-o-o-o-o-o-+o+ Figure 3. Enzymatic debranching of glycogen as mediated by amylo-M-glucosidase, transferase (oligo-l,4 + 1,4-glucantransferase)in coqjuction with phosphorylase. 0,glucose; a, reducing glucose unit; -, 1,4 bond; +, 1.6 bond; 0 , a-1,bglucose. Adopted from reference 53.

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The branching enzyme catalyzes the synthesis of the 1,6 linkages by a transfer reaction (52). When the outer chains are lengthened by glycogen synthase, they become sufficiently long (8-1 1 residues) for the branching enzyme to exchange an existing 1,4 linkage for a new 1,6 linkage (Fig. 4). The action of these two enzymes is separate but presumably coordinated with the enzymes that act upon the 1,4 linkages. The overall enzymatic pathway for glycogen metabolism is now well understood. As shown in Fig. 5 , UDPG is synthesized from glucose-l-phosphate and UTP by the enzyme UDPG pyrophosphorylase with inorganic pyrophosphate (PPi) as the other reaction product. Uridine diphosphoglucose is then used as the direct glucosy1 donor by the rate-determining enzyme of synthesis, glycogen synthase, to elongate the outer chains of glycogen. The branching enzyme amylo-1,4 + 1,6-transglucosylase then inserts the branch points by the transfer reaction already discussed. In glycogen degradation, the chief rate-determining enzyme is

Figure 4. Action of branching enzyme, amylo-14 + 1,6-transglucosylase.0 , a-l,4linked glucose; 0 , a-1,Clinked glucose.

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JOSEPH LARNER

Synthesis

Degradation

H

Clvmonn,

/

/

enzyme

Walker-Whelan dextrin (symmetric)

Elongated out& chain glycogen

t

Glycogen1

Debranching enzyme Glycogen synthase

Cori-Larner dextrin (asymmetric) Amylo- 1, 6glucosidase Glucose

UDPG L PPi

Glycogen with outer branches removed UDPG pyro-

UPT

Phosphorylase

Glucose- 1- P

Figure 5. The glycogen cycle. The enzymes that catalyze the synthesis and degradation of the a-1,4 and a,-1,6 linkages are shown. From Lamer, J., in Enryrne Therapy in Genetic Diseases IX,Birth Defects, Original Article Series, Bergsma, D . , Ed., Williams and Wilkins, Baltimore, 1973, p. 149.

phosphorylase. Although catalyzing a reversible reaction in vitro, in the cell, it is driven toward degradation by the high millimolar concentration of Pi (3-5 mM)compared to the very low micromolar concentration of glucose-1-phosphate (10-20 JAM)(55). At phosphorylase equilibrium, the ratio of Pi/glucose-1-phosphate is -3 : 1, but in the cell the ratio is 300: 1 (55). Finally, the debranching enzyme, amylo-1-4 + 1,4-transferase/amylo-l,&glucosidase rearranges the symmetric phosphorylase limit dextrin (Fig. 3) to the asymmetric limit dextrin and then cleaves the exposed a-1,dlinked glucose residue to free glucose. Thus, during metabolic degradation of glycogen by the two enzymes, phosphorylase and debranching enzyme, over 90% glucose-1-phosphate and