David H. Brown

The isolation of pyridoxal-5-phosphate from crystalline muscle phosphorylase

Abstract Dialyzed and Norit-treated muscle phosphorylase a, after recrystallization from versene-glycerophosphate, contains 8 organic phosphate groups per mole or 2 phosphate groups per subunit of molecular weight of 125,000. Four of these phosphate groups are extracted by precipitation of the enzyme with trichloroacetic or perchloric acid. The extracted phosphate compound was isolated as the barium salt and identified as pyridoxal-5-phosphate by its spectrum and by specific enzymic tests. Column chromatography of the trichloroacetic acid extract and paper electrophoresis did not reveal the presence of other phosphorylated compounds. In particular, pyridoxamine-5-phosphate, adenylic acid and other nucleotides could not be detected. Free pyridoxal, pyridoxamine or pyridoxine were also absent.

The phosphorylation of D-(+)-glucosamine by crystalline yeast hexokinase

d-glucosamine has been shown to be converted to a phosphorylated amino sugar by ATP in the presence of crystalline yeast hexokinase and magnesium ions. The rate of the reaction is approximately that observed for d-glucose. The turnover number of the enzyme for glucosamine is 12,000 moles/105 grams hexokinase/minute at pH 7.8 and 30°. The enzymatically synthesized ester of glucosamine was isolated as the barium salt and shown to have full reducing power and to have an acid-stable phosphate group. The ester consumes four moles of sodium periodate per mole of compound. These facts, together with its elemental composition, show that it is d-glucosamine-6-phosphate. Aqueous solutions of the ester are not stable; with time, one or more substances are formed which have characteristic absorption spectra; simultaneously, the reducing power disappears.

[88] Enzymes of glycogen debranching: Amylo-1,6-glucosidase (I) and oligo-1,4→1,4-glucantransferase (II)

Publisher Summary This chapter describes the specific oligosaccharide substrates for the separate measurement of each enzymatic activity (I and II). The nature of the reaction catalyzed by II is demonstrated using these substrates. Enzymes I and II act with phosphorylase to bring about the total degradation of glycogen to glucose 1-phosphate and glucose. The amylo-l,6-glucosidase appears to act directly on a polysaccharide limit dextrin to form glucose from its outermost branch points. The separate activity of amylo-l,6-glucosidase (I) is measured with certainty only when the substrate used is a branched oligosaccharide with the general structural features of B 5 . A limit dextrin (LD) of glycogen may not be a specific substrate for (I), as the number of exposed branch point glucose residues in the LD is not known with certainty. The initial rate of glucose formation from an LD may depend only on the action of (I) and be independent of the prior action of (II). The enzymatic activity of Oligo-l,4 → 1,4-glucantransferase (II) consists of the transfer of terminal maltosyl and, to a greater extent, maltotriosyl residues from α-l,4-1inkage in one chain to α-l,4-1inkage in another. The reagents used, procedure followed, and the steps involved in the purification are also described in the chapter.

The subcellular distribution of enzymes in type II glycogenosis and the occurrence of an oligo-α-1,4-glucan glucohydrolase in human tissues

Summary Biochemical analyses of the tissues from a case (B.J.P.) of Type II glycogenosis revealed generalized storage of glycogen of normal structure, the lack of an α-glucosidase active at pH 4.5, and the presence of all other enzymes whose deficiencies have been implicated in other types of glycogenosis. Differential centrifugation of a sucrose homogenate of an unfrozen sample of liver taken by biopsy from this case showed that more than 50% of the activities of glucose-6-phosphatase (EC 3.1.3.9), uridine diphosphoglucose—α-glucan transghico-sylase (EC 2.4.1.11), α-amylase (EC 3.2.1.1), and of an α-glucosidase active at neutral pH were recovered in the 100 000 × g pellet which also contained most of the glycogen of the tissue. Investigation of the substrate specificity of the α-glucosidase at pH 7.1 showed that it was a hitherto unrecognized type of oligo-α-1,4-glucan glucohydrolase. This enzyme formed no glucose from glycogen but acted rapidly on oligosaccharides (maltose and maltotriaose) to yield glucose. The enzyme has been found in biopsy and autopsy liver samples from ten individuals and in nineteen skeletal muscle samples representative of a vaiiety of types of glycogen storage disease.

[67] α-1,4-glucan: α-1,4-glucan 6-glycosyltransferase from mammalian muscle

Publisher Summary This chapter discusses the synthesis of α-1,4-glucan 6-glycosyltransferase from mammalian muscle. The assay depends on the increase in the rate of formation of polysaccharide from G-1-P by phosphorylase on addition of the branching enzyme. In the absence of branching enzyme, long amylase chains are formed by de novo synthesis. This reaction is extremely slow, as the concentration of end groups with which phosphorylase reacts remains very low. In the combined enzyme system the increased number of end groups that arise as a result of the branching of the growing polysaccharide chain allows the phosphorylase reaction to proceed at a faster rate. In this system, the formation of orthophosphate is an indirect measure of branching enzyme action. In an alternate assay procedure, the decrease in optical density of the iodine complex of corn amylopectin is described by Larner as an assay for the action of the branching enzyme from rat liver. The branching enzyme acts slowly on long linear chains of α-l,4-1inked glucose units. It is found that while liver glycogen does not serve as a substrate for branching, amylose and the limit dextrin of amylopectin prepared by β-amylase can serve as substrates.

The molecular heterogeneity of purified human liver lysosomal α-Glucosidase (acid α-Glucosidase)

Abstract An α-glucosidase active at acid pH and presumably lysosomal in origin has been purified from human liver removed at autopsy. The enzyme has both α-1,4-glucosidase and α-1,6-glucosidase activities. The K m of maltose for the enzyme is 8.9 m m at the optimal pH of 4.0. The K m of glycogen at the optimal pH of 4.5 is 2.5% (9.62 m m outerchain end groups). Isomaltose has a K m of 33 m m when α-1,6-glucosidase activity is tested at pH 4.2. The enzyme exists in several active charge isomer forms which have p I values between 4.4 and 4.7. These forms do not differ in their specific activities. Electrophoresis in polyacrylamide gels under denaturing conditions indicates that the protein is composed of two subunits whose approximate molecular weights are 88,000 and 76,000. An estimated molecular weight of 110,000 was obtained by nondenaturing polyacrylamide gel electrophoresis. When the protein was chromatographed on Bio-Gel P-200 it was separated into two partially resolved active peaks which did not differ in their charge isomer constitution or in subunit molecular weights. One peak gave a strongly positive reaction for carbohydrate by the periodic acid-Schiff method and the other did not. Both had the same specific activity. The enzyme was antigenic in rabbits, and the antibodies so obtained could totally inhibit the hydrolytic action of the enzyme on glycogen but were markedly less effective in inhibiting activity toward isomaltose and especially toward maltose. Using these antibodies it was found that liver and skeletal muscle samples from patients with the “infantile” form or with the “adult” form of Type II glycogen storage disease, all of whom lack the lysosomal α-glucosidase, do not have altered, enzymatically inactive proteins which are immunologically cross-reactive with antibodies for the α-glucosidase of normal human liver.

CHAPTER 5 – Glycogen-Storage Diseases*: Types I, III, IV, V, VII and Unclassified Glycogenoses

Publisher Summary This chapter discusses glycogen-storage diseases and some of the rarer types in which still other enzymic deficiencies have been implicated. Hepatic glucose 6-phosphatase is a microsomal enzyme with an optimum for activity between pH 6 and 7. Children lacking glucose 6-phosphatase are subject to hypoglycemia but frequently are asymptomatic at extremely low blood-sugar levels. In Type III glycogenosis, a polysaccharide accumulates whose structure resembles that of the limit dextrin produced by the degradation of glycogen by phosphorylase. As in Type I glycogenosis, the infant with limit dextrinosis has hepatomegaly, may be hypoglycemic, and when in the fasting state, fails to show a glucemic response to epinephrine or glucagon. The four most commonly encountered types of glycogen-storage disease are Types I, II, III, and VI, where VI is characterized by polysaccharide storage in the liver (and no evidence for muscle involvement) with no known enzymic cause.

Preparation and properties of the glycogen-debranching enzyme from rabbit liver

Abstract The glycogen-debranching enzyme, oligo-α-1,4-glucan: α-1,4-glucan-4-glycosyl-transferase-amylo-1,6-glucosidase has been purified about 500-fold from rabbit liver. The enzyme preparation has been shown to have both transferase and glucosidase activities. In view of the fact that the protein moves as one band in discgel electrophoresis and possesses the two activities in constant ratio throughout its purification, it is concluded that one protein in fact possesses both activities. The molecular weight of the enzyme has been found to be about 179 000 by sucrose-density gradient centrifugation. The enzyme is strongly inhibited when assayed in Tris buffer and somewhat less so in imidazole buffer as compared with citrate or phosphate buffers in which the optimum for activity is at about pH 6 when a limit dextrin of glycogen, prepared by prior phosphorylase action, is the substrate. Enzyme activity is also reversibly inhibited by urea at concentrations below 2 M. Guanidine at 0.15 M produces 50% inhibition of glucose formation from a phosphorylase limit dextrin.

The experimental production of glycogen storage in cultured human fibroblasts

Summary When normal human fibroblasts are cultured in the presence of D-(+)-trehalose added to a complete medium containing calf serum, the cells contain more glycogen than control cells grown in the absence of this disaccharide. Trehalose has been found to be a non-competitive inhibitor (KI = 5 to 8 mM) of the fibroblast α-glucosidase when this enzyme acts to degrade glycogen in vitro at pH 4.2. By growing normal cells in the presence of the non-metabolizable disaccharide, the half-life of their glycogen can be increased until its rate of turnover approaches that which is characteristic of glycogen in fibroblast cell lines from patients with Type II glycogen storage disease in which the α-glucosidase active at pH 4 is absent.

Phosphorylase and uridinediphosphoglucose-glycogen transferase in pyridoxine deficiency

Abstract The total phosphorylase activity of the skeletal muscle of rats maintained on a pyridoxine deficient diet has been found to fall to 35% of the normal value. The phosphorylase a activity of the tissue of these rats has the normal value, and the glycogen content of the muscles of such deficient rats is not different from that of control animals. The apparent activity of uridine diphosphoglucose-glycogen transferase is not changed from the normal level in pyridoxine deficiency.