The initiation of glycogen biosynthesis in rat heart. Studies with a purified preparation

Two fractions of glycogen synthase were isolated from rat cardiac muscle on the basis of a different affinity for DEAE-cellulose and omega-aminobutyl-agarose. One of these fractions was able to transfer glucosyl residues from UDP-glucose not only to glycogen (GS-1 activity) but also to an endogenous acceptor. The latter reaction (GS-2 activity) occurred in the absence of added glycogen, and its reaction product was insoluble in trichloroacetic acid. This compound was degraded by amylolytic enzymes, thus showing that the product synthesized on the endogenous acceptor was an alpha 1,4-glucan. After incubation with alpha-amylase-free proteolytic enzyme, the compound was rendered trichloroacetic acid-soluble. Polyacrylamide gel electrophoresis, under both native and denaturing conditions, showed that GS-2 reaction products moved electrophoretically associated to protein. Our results give further evidence for the association between an alpha 1,4-glucan and protein, which we postulate is related to the initiation of glycogen biosynthesis.     

Characterization of a protein:glucosyltransferase activity in human platelets

Human platelets exhibited significant glucosyltransferase activity, that transfer [14C]glucose from UDP-Glc to an endogenous protein acceptor. The enzyme protein:glucosyltransferase responsible for the catalysis was characterized and compared with glycogen:glucosyltransferase. We describe a partial separation of both activities, the ratio of protein:glucosyltransferase/glycogen:glucosyltransferase varied from 7:1 in a crude homogenate of platelets to 36:1 in the Sephadex G-100 column. This procedure failed to separate the protein:glucosyltransferase from its endogenous acceptor. Glucosylation of protein demonstrated dependence with respect to time and both protein and UDP-Glc concentration, and was saturated by very low concentration of donor and acceptor substrates. It was inhibited 76% by 5 mM Mn2+ concentration and was activated 23 and 11% by 5 mM concentrations of Ca2+ and Mg2+, respectively. With respect to glycogen:glucosyltransferase, when the effect of time, protein, and substrate concentration were determined under identical conditions, it did not show the same dependence. At 5 mM concentration, Mn2+, Ca2+, and Mg2+ were activators of the enzyme 43, 80, and 200%, respectively. On the basis of these characteristics, we conclude that the synthesis of glucoprotein and glycogen are catalyzed by two distinct enzymes. Addition of exogenous glycogen (range 0.002-1%) inhibited the protein:glucosyltransferase, whereas at 0.001-0.007% concentration it was acceptor substrate for glycogen:glucosyltransferase activity. At higher concentrations this activity was strongly inhibited. The concentration of glycogen in platelets could play a regulatory role in forming the glucoprotein and the glycogen molecules.     

Factors affecting glucosyl and mannosyl transfer to dolichyl monophosphate by liver cell-free preparations

GDP-mannose and UDP-mannose (each at less than 1 micrometer) markedly inhibit glucosyl transfer from UDP-glucose (1.6 micrometer( to dolichyl phosphate in liver microsomal preparations. The biphasic response suggests the presence of two glucosyl transferases only one of which is inhibited. The inhibition appears to be a property of the intact nucleotide phosphate sugars and not due to competition for a limited pool of dolichyl phosphate. UDP-galactose and UDP-xylose cause a less marked inhibition of the same enzyme. The failure of UDP-glucose to inhibit mannosyl transfer suggests that the pool of dolichol monophosphate used by mannosyl transferase is not available to the glucosyl transferase. The relationship between the degree to which an exogenous prenol phosphate acts as an acceptor of mannose and the degree to which it inhibits mannosylation of endogenous dolichyl monophosphate varies among different prenyl phosphates. Mannosyl transferase exhibits two pH optima.     

Studies on glucosyltransferase and endogenous glucosyl acceptor in Bacillus cereus AHU 1030 membranes

A glucosyltransferase, extracted from the membranes of Bacillus cereus AHU 1030 with Tris-HCl buffer containing 0.1% Triton X-100 at pH 9.5, was separated from an endogenous glucosyl acceptor by chromatography on DEAE-Sepharose CL-6B subsequent to chromatography on Sepharose 6B. Structural analysis data showed that the glucosyl acceptor was a glycerol phosphate polymer linked to beta-gentiobiosyl diglyceride. The enzyme catalyzed the transfer of glucosyl residues from UDP-glucose to C-2 of the glycerol residues of repeating units of the acceptor. On the other hand, a lipoteichoic acid which contained 0.3 D-alanine residue per phosphorus was isolated from the cells by phenol treatment at pH 4.6. Except for the presence of D-alanine, this lipoteichoic acid had the same structure as the glucosyl acceptor. The rate of glucosylation observed with the D-alanine-containing lipoteichoic acid as the substrate was less than 40% of that observed with the D-alanine-free lipoteichoic acid, indicating that the ester-linked D-alanine in the lipoteichoic acid interferes with the action of the glucosyltransferase. The enzyme also catalyzed glucosylation of poly(glycerol phosphate) which was synthesized in the reaction of a separate enzyme fraction with CDP-glycerol. Thus, it is likely that the glucosyltransferase functions in the synthesis of cell wall teichoic acid.     

Amylopectin Synthase of Eimeria tenella: Identification and Kinetic Characterization

ABSTRACT. A soluble enzyme amylopectin synthase (UDP-glucose-α 1,4-glucan α-4-glucosyltransferase) which transfers glucose from uridine 5'-diphosphate glucose (UDP-glucose) to a primer to form α-I,4-glucosyl linkages has been identified in the extracts of unsporulated oocysts of Eimeria tenella . UDP-glucose and not ADP-glucose was the most active glucosyl donor. Corn amylopectin, rabbit liver glycogen, oyster glycogen and corn starch served as primers; the latter two were less efficient. The enzyme has an apparent pH optimum of 7.5 and exhibited typical Michaelis-Menten kinetics with dependence on both the primer and substrate concentrations. The Michaelis constants (Km). with respect to UDP-glucose, was 0.5 mM; and 0.25 mg/ml and 1.25 mg/ml with respect to amylopectin and rabbit liver glycogen. The product formed by the reaction was predominantly a glucan containing α-1,4 linkages. The specificity of the enzyme suggests that this enzyme is similar to glycogen synthase in eukaryotes and has been designated as amylopectin synthase (UDP-glucose-α-1,4-glucosetransferase EC 2.4.1.11).     

Evidence for multiple enzymes in the dolichol utilizing pathway of glycoprotein biosynthesis.

A comparison has been made of the enzymes catalyzing the transfer of mannose, glucose and N-acetylglucosamine from, respectively, GDPmannose, UDP-glucose and UDP-N-acetylglucosamine to endogenous dolichol phosphate (Dol-P) in liver Golgi membranes. Evidence is presented with suggests that all three reactions utilize the same pool of Dol-P. The transfer of mannose from GDP-Man to Dol-P is not inhibited by 0.1 mM UDP or UMP; 0.1 mM GDP did block the accumulation of mannose in Dol-P-Man. The net transfer of glucose and N-acetylglucosamine to Dol-P is prevented by 0.1 mM UDP but not 0.1 mM GDP. UDPglucose inhibits the reverse of the glucose transfer reaction but not the reverse of the N-acetylglucosamine or mannose trasfer reaction. On the basis of this, and other data, it is concluded that the three sugar transfer reactions utilize separate enzymes.     

alpha-Glucan synthesis on a protein primer, uridine diphosphoglucose: protein transglucosylase I. Separation from starch synthetase and phosphorylase and a study of its properties

It was found that the DEAE-cellulose-treated UDP-Glc:protein transglucosylase I catalyzing the first step (reaction 1) in the formation of alpha-glucan bound to protein in potato tuber is not only specific for the glucosyl donor but also for the endogenous acceptor. A single radioactive 38-kDa macromolecular component appeared during denaturing polyacrylamide gel electrophoresis of reaction 1 product. The labeled component is probably the polypeptide subunit of the endogenous acceptor which is being glucosylated. The radioactivity incorporated in reaction 1 product was isolated from a protease digest as a low-molecular-mass glucopeptide fraction. A beta-elimination reaction carried out in the presence of a reducing agent demonstrated that only one glucosyl moiety is transferred from UDP-Glc to the aminoacyl residue, thus forming an O-glucosidic linkage. 3H-labeled sodium borohydride showed that serine and threonine are involved in the peptide bond to glucose. Ion-exchange chromatography on DEAE-cellulose, affinity chromatography on concanavalin-A--Sepharose, gel filtration on Sephacryl S-300 and sucrose density gradient centrifugation failed to separate the enzyme catalyzing reaction 1 from the endogenous acceptor.     

Manganese sulfate-dependent glycosylation of endogenous glycoproteins in human skeletal muscle is catalyzed by a nonglucose 6-P-dependent glycogen synthase and not glycogenin

Glycogenin, a Mn2+-dependent, self-glucosylating protein, is considered to catalyze the initial glucosyl transfer steps in glycogen biogenesis. To study the physiologic significance of this enzyme, measurements of glycogenin mediated glucose transfer to endogenous trichloroacetic acid precipitable material (protein-bound glycogen, i.e., glycoproteins) in human skeletal muscle were attempted. Although glycogenin protein was detected in muscle extracts, activity was not, even after exercise that resulted in marked glycogen depletion. Instead, a MnSO4-dependent glucose transfer to glycoproteins, inhibited by glycogen and UDP-pyridoxal (which do not affect glycogenin), and unaffected by CDP (a potent inhibitor of glycogenin), was consistently detected. MnSO4-dependent activity increased in concert with glycogen synthase fractional activity after prolonged exercise, and the MnSO4-dependent enzyme stimulated glucosylation of glycoproteins with molecular masses lower than those glucosylated by glucose 6-P-dependent glycogen synthase. Addition of purified glucose 6-P-dependent glycogen synthase to the muscle extract did not affect MnSO4-dependent glucose transfer, whereas glycogen synthase antibody completely abolished MnSO4-dependent activity. It is concluded that: (1) MnSO4-dependent glucose transfer to glycoproteins is catalyzed by a nonglucose 6-P-dependent form of glycogen synthase; (2) MnSO4-dependent glycogen synthase has a greater affinity for low molecular mass glycoproteins and may thus play a more important role than glucose 6-P-dependent glycogen synthase in the initial stages of glycogen biogenesis; and (3) glycogenin is generally inactive in human muscle in vivo.     

Action of degradative enzymes on the light fraction of bovine septa protein polysaccharide

1. The administration of cortisol and of other glucocorticoid steroids to starved mice produced an increase in liver glycogen content, an elevation of glycogen-synthetase activity and a predominantly particulate localization of both phosphorylase and glycogen-synthetase enzymes. 2. Three daily doses of actinomycin D caused a marked glycogen depletion, a significant decrease in glycogen-synthetase activity, the solubilization of phosphorylase and glycogen synthetase and the following effects on the activities of various other enzymes: a decrease in UDP-glucose pyrophosphorylase and phosphoglucomutase, an increase in glucose 6-phosphate dehydrogenase and no change in glucose 6-phosphatase, 6-phosphogluconate dehydrogenase, pyruvate kinase and UDP-glucose dehydrogenase. 3. Glucose ingestion, but not cortisol administration, reversed the effects of actinomycin D on liver glycogen content and on the activities of phosphorylase and glycogen synthetase.     

Properties of glucosyltransferase and glucan transferase from spinach

A glucosyl and a glucosyl-glucan transferase activity from spinach (Spinacia oleracea L. var. Matador) leaves have been partially purified and characterized. The latter activity (fraction 1 after diethylaminoethylcellulose chromatography) is responsible for the transfer of glucosyl as well as of maltosyl, maltotriosyl, and higher homologous residues to glucose giving rise to maltose and the correspondingly larger molecules. This fraction also shows β-amylase activity. The transfer takes place only to glucose; maltose, as well as other α-1,4-glucans, serve as donors. The enzyme fraction 2 is amylase-free and catalyzes only the transfer of glucosyl moieties, again with high acceptor specificity to glucose. Maltose and larger α-1, 4-glucans, with the exception of maltotriose and maltotetraose, act as donors. The physiological function of these enzymes may be the formation of oligosaccharide primers for starch synthetase or phosphorylase.     

Structural basis for subunit assembly in UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae

UDP-glucose is the universal activated form of glucose, employed in all organisms for glucosyl transfer reactions and as precursor for various activated carbohydrates. In animal and fungal metabolism, UDP-glucose is required for utilization of galactose and for the synthesis of glycogen, the major carbohydrate storage polymer. The formation of UDP-glucose is catalyzed by UDP-glucose pyrophosphorylase (UGPase), which is highly conserved among eukaryotes. Here, we present the crystal structure of yeast UGPase, Ugp1p. Both in solution and in the crystal, Ugp1p forms homooctamers, which represent the enzymatically active form of the protein. Ugp1p subunits consist of three domains, with the active site presumably located in the central SpsA GnT I core (SGC) domain. The association in the octamer is mediated by contacts between left-handed beta-helices in the C-terminal domains, forming a toroidal solenoid structure in the core of the complex. The catalytic domains attached to this scaffold core do not directly contact each other, consistent with simple Michaelis-Menten kinetics found for Ugp1p. Conservation of hydrophobic residues at the subunit interfaces suggests that all fungal and animal homologs form this quarternary structure arrangement in contrast to monomeric plant UGPases, which have charged residues at these positions. Implications of this oligomeric arrangement for regulation of UGPase activity in fungi and animals are discussed.     

Solubilization and properties of mannose and N-acetylglucosamine transferases involved in formation of polyprenyl-sugar intermediates.

A particulate fraction from porcine aorta catalyzed the incorporation of N-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc into both GlcNAc-pyrophosphorylpolyprenol and GlcNAc-GlcNAc-pyrophosphorylpolyprenol. This transfer utilized endogenous lipid and required a divalent cation. Mn2+ was the best metal ion and was optimum at 2.3 mM. This same particulate fraction was previously shown to transfer mannose from GDP-[14C]mannose to endogenous lipid to form mannosylphosphorylpolyprenol (Chambers, J., and Elbein, A.D. (1975) J. Biol. Chem. 250, 6904-6915). Both the GlcNAc activities and the mannose activity were solubilized by treatment of the particulate fraction with the detergent Nonidet P-40. The enzymes were partially purified by chromatography on DEAE-cellulose and on Sephadex G-200. These soluble enzymes required the addition of acceptor lipid for activity. An acidic lipid fraction, isolated from pig liver and having the properties of dolichyl phosphate, was active with either the GlcNAc or the mannose transferase. Chemically synthesized dolichyl phosphate was also active with either of these enzymes. The products formed from either GlcNAc or mannose by the soluble transferases were similar to those formed by the particulate enzyme. Thus the major product formed from UDP-[3H]GlcNAc was GlcNAc-pyrophosphoryldolichol with small amounts of the disaccharide-lipid while the product formed from GDP-[14C]mannose was mannosylphosphoryldolichol.     

Function of alpha-D-glucosyl monophosphorylpolyprenol in biosynthesis of cell wall teichoic acids in Bacillus coagulans.

D-[alpha-14C]]glucosyl phosphorylpolyprenol ([ 14C]Glc-P-prenol) was formed from UDP-D-[14C]glucose in each of the membrane systems obtained from Bacillus coagulans AHU 1631 and AHU 1634 and two Bacillus megaterium strains. Membranes of these B. coagulans strains, which possess beta-D-glucosyl branches on the repeating units in their major cell wall teichoic acids, were shown to catalyze the transfer of the glucose residue from [14C]Glc-P-prenol to endogenous polymer. On the other hand, membranes of B. coagulans AHU 1366, which has no glucose substituents in the cell wall teichoic acid, exhibited neither [14C]Glc-P-prenol synthetase activity nor the activity of transferring glucose from [14C]Glc-P-prenol to endogenous acceptor. The enzyme which catalyzes the polymer glycosylation in the former two B. coagulans strains was most active at pH 5.5 and in the presence of the Mg2+ ion. The apparent Km for [14C]Glc-P-prenol was 0.6 microM. Hydrogen fluoride hydrolysis of the [14C]glucose-linked polymer product yielded a major fragment identical to D-galactosyl-alpha(1----2)(D-glucosyl-beta(1----1/3)) glycerol, the dephosphorylated repeating unit in the major cell wall teichoic acids of these B. coagulans strains. This result, together with the behavior of the radioactive polymer in chromatography on Sepharose CL-6B, DEAE-Sephacel, and Octyl-Sepharose CL-4B, led to the conclusion that [14C]Glc-P-prenol serves as an intermediate in the formation of beta-D-glucosyl branches on the polymer chains of cell wall teichoic acids in B. coagulans.     

Purification to homogeneity and characterization of a 1,3-beta-glucan (callose) synthase from germinating Arachis hypogaea cotyledons.

A 1,3-beta-D-glucan (callose) synthase (CS) from a plasma membrane fraction of germinating peanut (Arachis hypogaea L.) cotyledons has been purified to apparent homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino-terminal analysis, and the Western blots pattern. The purification protocol involved preparation of a high specific activity plasma membrane fraction, selective solubilization of the enzyme from the membrane with 0.5% digitonin at a protein-to-detergent ratio of 1:6, sucrose gradient centrifugation, and chromatography on hydroxylapatite and DEAE-Sephadex A-50. The purified CS shows a molecular mass of approximately 48,000 by SDS-PAGE, pH optimum of 7.4, leucine as the amino-terminal residue, Km for UDP-glucose of 0.67 mM, and Vmax of 6.25 mumol/min/mg protein. The enzyme is specific for UDP-glucose as the glucosyl donor and required Ca2+, at an optimum concentration of 2-5 mM, for activity. The enzyme activity was inhibited by nucleotides (ATP, GTP, CTP, UTP, UDP, and UMP). The enzyme activity was also inhibited by the addition of EDTA or EGTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The reaction product formed during incubation of UDP-[14C]glucose and cellobiose with purified enzymes was susceptible to digestion by exo-(1,3)-beta-glucanase, but was resistant to alpha- and beta-amylases and to periodate oxidation, indicating that the polymer formed was 1,3-beta-glucan, and beta-1,4 and beta-1,6 linkages were absent.     

Dodecyl-{beta}-D-maltoside as a substrate for glucosyl and xylosyl transfer by glycogenin

Glycogenin is the core protein of glycogen proteoglycan andis, at the same time, a self-glucosylating enzyme which catalysesearly glucosyl transfer steps in the biosynthesis of glycogen.In the course of this process, glycogenin is glucosylated progressivelyuntil an oligosaccharide containing 8–11 glucose residueshas been formed. Although glycogenin, under physiological conditions,is both enzyme and acceptor in the glucosyl transfer reactions,it is also capable of utilizing p-nitrophenyl-linked malto-oligosaccharidesas exogenous acceptors. In view of the potential usefulnessof exogenous acceptors in the study of the mechanism of theglycogenin reaction, we have expanded the search for such compoundsand report here that several alkyl glucosides and alkyl maltosidesmay serve as acceptors in glucosyl transfer by beef kidney glycogenin.Dodecyl-ß-D-maltoside (K_m {small tilde}100 µM)was the most effective acceptor among the compounds tested andyielded 30 times as much product as p-nitrophenyl-     

Galactosylation of endogenous proteins from human platelets

Human platelets have been shown to contain the enzyme glycoprotein:galactosyltransferase that catalyzes the transfer of galactose to an endogenous protein acceptor present in the platelet. Galactosylation of added ovalbumin also occurs. The activity was extracted with 30 mM Tris buffer (pH 7.5). The endogenous activity was enriched 1.4-fold (compared with the crude homogenate) in the fraction, 105,000 g pellet, and the exogenous enzyme was retained in the respective supernatant. The two galactosyltransferase activities showed proportionality to time, protein, and substrate concentration, and were identical in pH dependence and Mn+2 requirement. The effect of Triton X-100 (range 0-1.5%) in the assay system appeared to be different for both activities: with the optimum concentration of detergent (0.15%) the endogenous activity increased by 50% whereas the exogenous activity was augmented 5-fold. From a number of sugar nucleotides tested as glycosyl donor into the endogenous proteins, the optimum substrate was UDP-Glc (100%), followed by UDP-Gal (80%), GDP-Man (24%), UDP-Glc-NAc (21%), UDP-Xyl (19%), and ADP-Glc (5%). An appropriate exogenous acceptor for UDP-Glc as donor was not found. The different solubilization of galactosyl- and glucosyltransferase activities by Triton X-100 suggests that they are distinct enzymes. In addition, the exogenous galactosyltransferase activity achieved after the treatment was much higher (940%) than the endogenous (26%). It is suggested that these differences on both galactosyltransferases could reflect changes in the accessibility of the exogenous substrate to the enzyme.     

Biosynthesis of Glycogen and Starch in Cryptococcus laurentii

Cells of Cryptococcus laurentii, when grown in liquid culture on 2% glucose close to neutral pH, showed glycogen granules throughout the cytoplasm. Glycogen levels of C. laurentii cells reached maximal levels just before onset of stationary phase. Concomitantly, a sharp rise in total and specific activity of glycogen synthetase was observed. Conversely, glycogen phosphorylase reached its highest specific activity approximately 3 hr after the glycogen peaked and remained high until most of the endogenous glycogen was utilized. Uridine diphosphoglucose pyrophosphorylase activity was always an order of magnitude higher than glycogen synthetase during log phase, but fell off rapidly after the cells reached stationary growth. Kinetic properties of the glycogen synthetase showed that the enzyme is always activated by glucose-6-phosphate, although the degree of activation by glucose-6-phosphate was found to be somewhat variable. The accelerated uptake of glucose commencing with the onset of stationary phase is explained by the rapid formation of extracellular acidic polysaccharide, which continues as long as there is glucose in the medium. In cells grown at pH 3.4, where no detectable extracellular acidic polysaccharide was formed, glucose uptake drastically declined when the cells reached stationary phase. These cells also contained glycogen-like granules in the cytoplasm. The evidence presented indicates that these granules are in fact glycogen, and that its structure does not resemble that of the starch excreted by cells grown at acidic pH.     

Regulation of basal and insulin-stimulated glycogen synthesis in cultured hepatocytes. Inverse relationship to glycogen content

Cultured rat hepatocytes were used to characterize the relationship between cellular glycogen content and the basal rate, as well as response to insulin of glycogen synthesis. Depending on the concentration of medium glucose, glycogen-depleted monolayers accumulated glycogen between 24 and 48 h of culture up to the fed in vivo level. Insulin at 100 nM stimulated glycogen deposition 20-fold at 1 mM and 1.5-fold at 50 mM glucose. The rate of further glycogen storage decreased with time and increasing glycogen content. In hepatocytes preincubated with 1-50 mM glucose during 24-48 h, short-term basal and insulin-dependent incorporation of 10 mM [14C]glucose into glycogen was inversely related to the actual cellular glycogen content. This was not due to different intracellular dilution of the label, since the specific radioactivity of UDP-glucose was similar in all groups. 125I-Insulin binding indicated that insulin receptors were also not involved in this phenomenon. An inverse relationship was also found between glycogen content and the stimulation of glycogen synthase I activity by insulin, whereas the basal activity of the enzyme was dissociated from the rate of incorporation of [14C]glucose. Basal net glycogen deposition at 10 mM glucose was also inversely related to cellular glycogen; however, no such relation was evident in the presence of insulin due to the overlapping inhibition of glycogenolysis. These studies suggest that the glycogen-mediated inhibition of the activation of glycogen synthase I is operative in the cultured hepatocyte and leads to an apparent inverse relationship between the actual glycogen content and basal as well as insulin-dependent glycogenesis.     

Rat adipose tissue glycogen synthase. Evidence for multiple discrete kinetic species and their interconversion.

Rat adipose tissue glycogen synthase has been kinetically characterized. The classical D form has an apparent Km for UDP-glucose of 0.7 mM and 0.4 mM in the absence and presence of glucose 6-phosphate, respectively. The apparent Ka for glucose 6-phosphate is 0.6 mM. The effect of glucose 6-phosphate on the D form is to enhance the Vmax 7-fold. The I form is also affected by glucose 6-phosphate (Ka, 0.025 mM) but the Vmax is increased only by 20%; apparent Km values for UDP-glucose are 0.4 mM and 0.045 mM in the absence and presence of glucose 6-phosphate, respectively. In addition, two new kinetically distinguishable forms have been observed. The first, designated glycogen synthase Q, arises from an Mg2+ATP-dependent deactivation of the I form. The apparent Km values of glycogen synthase Q for UDP-glucose are identical with those of the I form; however, the apparent Ka for glucose 6-phosphate (0.2 mM) is 8-fold higher than that for the I form and one-third that for the D form. Preparations from fasted or diabetic rats contain a form of glycogen synthase, designated glycogen synthase X, that has a much lower affinity for glucose 6-phosphate than the D form (apparent Ka, 3 mM); the apparent Km values for UDP-glucose are similar to those of the D form (0.7 mM and 0.3 mM in the absence and presence of glucose 6-phosphate, respectively). In preparations from fasted rats a stepwise Mg2+-dependent conversion was demonstrated of synthase X to D to Q to I; this sequential conversion was reversed on incubation with Mg2+ATP. In preparations from fed rats, synthase Q could be generated either by limited activation (from the D form) or, after conversion to the I form, by deactivation with Mg2+ATP. However, even prolonged incubation with Mg2+ATP failed to generate the D (or X) form.     

In vitro glucosylation of gibberellins

In maturing fruits ofPhaseolus coccineus a soluble glucosyltransferase activity occurs which converts gibberellins into their O-glucosides. The enzyme glucosylates GA₃ and structurally closely related gibberellins (GA₇ and GA₃₀) to their 3-O-glucosides by transfer of glucose preferentially from UDP-glucose. From cell suspension cultures ofLycopersicon peruvianum cytosolic glucosyltransferases were isolated which in the presence of UDP-glucose converted GA₇ and GA₉ to the corresponding glucosyl esters. In both cases numerous other gibberellins failed to serve as substrates. Thus, the enzymes are UDP-glucose: gibberellin glucosyltransferases of considerable substrate specificity.