Presence Of Glucose 6-phosphate Research Articles

Fabrication of a highly sensitive glucose-6-phosphate biosensor based on direct electron transfer using phage display and autodisplay technologies

Glucose in biological cells is metabolized and exists as glucose-6-phosphate (G6P). A G6P biosensor was fabricated using a combination of phage display and autodisplay technologies for the detection of G6P. Active G6P dehydrogenase (G6PDH) was expressed and anchored to the outer membrane of Escherichia coli (E. coli) cells using autodisplay technology. Autodisplayed G6PDH exhibited high activity as confirmed by OM protein analysis. A conductive nanomesh layer was formed using a phage display to enhance the direct electron transfer of the enzyme. G6PDH autodisplaying E. coli cells were then immobilized on the layer using a polyelectrolyte interlayer. The nicotinamide adenine dinucleotide phosphate redox pairs in the fabricated electrode of the autodisplayed G6PDH exhibited a redox reaction, resulting in a significant increase in the peak currents in the presence of G6P. The G6P biosensor exhibited a wide dynamic range with high sensitivity, exhibiting linear responses in the ranges of 0.1 fM – 10 nM and 10 nM – 100 μM. The biosensor detected G6P without interference from other sugar molecules. The combination of phage display and autodisplay technologies is a promising approach for the fabrication of highly sensitive and specific biosensors.

Insights into Glucose-6-phosphate Allosteric Activation of β-Glucosidase A.

Second-generation ethanol production involves the use of agricultural and forestry waste as feedstock, being an alternative to the first-generation technology as it relies on low-cost abundant residues and does not affect food agriculture. However, the success of second-generation biorefineries relies on energetically efficient processes and effective enzyme cocktails to convert cellulose into fermentable sugars. β-glucosidases catalyze the last step on the enzymatic hydrolysis of cellulose; however, they are often inhibited by glucose. Previous studies demonstrated that glucose-6-phosphate (G6P) is a positive allosteric modulator of Bacillus polymyxa β-glucosidase A, improving enzymatic efficiency, providing thermoresistance, and imparting glucose tolerance. However, the precise molecular details of G6P-β-glucosidase A interactions have not yet been described so far. We investigated the molecular details of G6P binding into B. polymyxa β-glucosidase A through in silico docking using the site identification by ligand competitive saturation technology followed by site-directed mutagenesis studies, from which an allosteric binding site for G6P was identified. In addition, a mechanistic shift toward the transglycosylation reaction as opposed to hydrolysis was observed in the presence of G6P, suggesting a new role of G6P allosteric modulation of the catalytic activity of β-glucosidase A.

Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate

Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

Revealing the important role of allosteric property in sucrose phosphate synthase from sugarcane with N-terminal domain deletion

Sucrose occupies many essential roles to control regulation of carbon partitioning in plants, including prokaryotic cells. Sucrose phosphate synthase (SPS; EC 2.4.1.14) is a key enzyme to catalyze the form of sucrose in primary sucrose synthesis pathway. Plants SPS has a molecular size around 120 kDa, which consists of N-terminal domain, C-terminal domain, and central domain. We produced the recombinant sugarcane SPS (SoSPS1) in Escherichia coli, however, the expression often appears to be a shorter form with retained enzyme activity. In our result, we reported that the shorter form is suggested to have a truncated N-terminal 20-kDa region. The truncated form of So SPS1 (ΔN-SPS) tends to enhance the specific activity 10-fold compared to full-length SoSPS1. The full-lenght SoSPS1 showed a remarkable allosteric activation by glucose-6-phosphate (G6P), while none of the N-terminal truncated form had such a characteristic. By kinetic analysis of full-length SoSPS1, a higher substrate affinity was shown in the presence of G6P. Conversely, the ΔN-SPS showed a similar substrate affinity whether G6P was added or not. Based on these results, we revealed that N-terminal region of SoSPS1 has essential role for allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity.

Liver-specific Inducible Nitric-oxide Synthase Expression Is Sufficient to Cause Hepatic Insulin Resistance and Mild Hyperglycemia in Mice

Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, plays an important role in obesity-induced insulin resistance. Inhibition of iNOS by gene disruption or pharmacological inhibitors reverses or ameliorates obesity-induced insulin resistance in skeletal muscle and liver in mice. It is unknown, however, whether increased expression of iNOS is sufficient to cause insulin resistance in vivo. To address this issue, we generated liver-specific iNOS transgenic (L-iNOS-Tg) mice, where expression of the transgene, iNOS, is regulated under mouse albumin promoter. L-iNOS-Tg mice exhibited mild hyperglycemia, hyperinsulinemia, insulin resistance, and impaired insulin-induced suppression of hepatic glucose output, as compared with wild type (WT) littermates. Insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and -2, and Akt was significantly attenuated in liver, but not in skeletal muscle, of L-iNOS-Tg mice relative to WT mice without changes in insulin receptor phosphorylation. Moreover, liver-specific iNOS expression abrogated insulin-stimulated phosphorylation of glycogen synthase kinase-3β, forkhead box O1, and mTOR (mammalian target of rapamycin), endogenous substrates of Akt, along with increased S-nitrosylation of Akt relative to WT mice. However, the expression of insulin receptor, IRS-1, IRS-2, Akt, glycogen synthase kinase-3β, forkhead box O1, protein-tyrosine phosphatase-1B, PTEN (phosphatase and tensin homolog), and p85 phosphatidylinositol 3-kinase was not altered by iNOS transgene. Hyperglycemia was associated with elevated glycogen phosphorylase activity and decreased glycogen synthase activity in the liver of L-iNOS-Tg mice, whereas phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and proliferator-activated receptor γ coactivator-1α expression were not altered. These results clearly indicate that selective expression of iNOS in liver causes hepatic insulin resistance along with deranged insulin signaling, leading to hyperglycemia and hyperinsulinemia. Our data highlight a critical role for iNOS in the development of hepatic insulin resistance and hyperglycemia.

Neurodegeneration and functional impairments associated with glycogen synthase accumulation in a mouse model of Lafora disease

Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin-positive (PV+) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV+ interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin–malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD.

The Crystal and Solution Studies of Glucosamine-6-phosphate Synthase from Candida albicans

Glucosamine 6-phosphate (GlcN-6-P) synthase is an ubiquitous enzyme that catalyses the first committed step in the reaction pathway that leads to formation of uridine 5′-diphospho- N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor of macromolecules that contain amino sugars. Despite sequence similarities, the enzyme in eukaryotes is tetrameric, whereas in prokaryotes it is a dimer. The activity of eukaryotic GlcN-6-P synthase (known as Gfa1p) is regulated by feedback inhibition by UDP-GlcNAc, the end product of the reaction pathway, whereas in prokaryotes the GlcN-6-P synthase (known as GlmS) is not regulated at the post-translational level. In bacteria and fungi the enzyme is essential for cell wall synthesis. In human the enzyme is a mediator of insulin resistance. For these reasons, Gfa1p is a target in anti-fungal chemotherapy and in therapeutics for type-2 diabetes. The crystal structure of the Gfa1p isomerase domain from Candida albicans has been analysed in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy- d-mannitol 6-phosphate (ADMP). A solution structure of the native Gfa1p has been deduced using small-angle X-ray scattering (SAXS). The tetrameric Gfa1p can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains. UDP-GlcNAc binds, together with a metal cation, in a well-defined pocket on the surface of the isomerase domain. The residues responsible for tetramerisation and for binding UDP-GlcNAc are conserved only among eukaryotic sequences. Comparison with the previously studied GlmS from E. coli reveals differences as well as similarities in the isomerase active site. This study of Gfa1p focuses on the features that distinguish it from the prokaryotic homologue in terms of quaternary structure, control of the enzymatic activity and details of the isomerase active site.

Polyamine enhances the regeneration of reduced glutathione by the activation of NADP-dependent dehydrogenases in yeast

Effect of polyamine on the regeneration of reduced glutathione (GSH) was analyzed in yeast. Spermine stimulated the reduction of oxidized glutathione in the presence of glucose 6-phosphate or isocitrate in permeabilized yeast cells. Stimulation of GSH formation was due to the enhancement of NADPH (reduced nicotinamide adenine dinucleotide phosphate) supply. Polyamine activated mitochondrial NADP (nicotinamide adenine dinucleotide phosphate)-isocitrate dehydrogenase (EC 1.1.1.42) and cytosolic glucose 6-phosphate dehydrogenase (EC 1.1.1.49) from yeast. Activating action of polyamine was largely the effect on the maximal velocity of these enzymes, and the order of effectiveness as the activators was spermine > spermidine > putrescine. The concentrations of spermine required for 50% activation of the enzymes were about 1—2 mM, values that were within the range of physiological concentrations of spermine. Polyamine can act as an antioxidant by stimulating the regeneration of reduced glutathione, the principal reactive oxygen scavenger, through the activation of cytosolic glucose 6-phosphate dehydrogenase and mitochondrial NADP-isocitrate dehydrogenase as the NADPH-generating enzymes in yeast.

Functional Characteristics of Hexokinase Bound to the Type A and Type B Sites of Bovine Brain Mitochondria

Hexokinase is released from Type A sites of brain mitochondria in the presence of glucose 6-phosphate (Glc-6-P); enzyme bound to Type B sites remains bound. Hexokinase of freshly isolated bovine brain mitochondria (Type A:Type B, ∼40:60) selectively uses intramitochondrial ATP as substrate and is relatively insensitive to the competitive (vs ATP) inhibitor and Glc-6-P analog, 1,5-anhydroglucitol 6-phosphate (1,5-AnG-6-P). After removal of hexokinase bound at Type A sites, the remaining enzyme, bound at Type B sites, does not show selectivity for intramitochondrial ATP and has increased sensitivity to 1,5-AnG-6-P. Thus, the properties of the enzyme bound at Type B sites are modified by removal of hexokinase bound at Type A sites. It is suggested that mechanisms for regulation of mitochondrial hexokinase activity, and thereby cerebral glycolytic metabolism, may depend on the ratio of TypeA:Type B sites, which varies in different species.

Expression of the pho regulon interferes with induction of the uhpT gene in Escherichia coli K-12.

The uptake of hexose 6-phosphates in Escherichia coli is mediated by the transporter UhpT, the synthesis of which is induced by the presence of glucose 6-phosphate (glucose 6-P) in the medium. Since this protein functions as an anion exchanger, it is generally assumed to be geared for the use of sugar phosphates as a carbon source. However, the question was unresolved whether this transporter can also provide the cells with glucose 6-P as a phosphate source. It is demonstrated in this work that UhpT-mediated glucose 6-P uptake does not allow the cells to grow on glucose 6-P as phosphate source. Hence, the expression of UhpT under phosphate limitation would not be particularly advantageous and some form of interaction between the uhp system and the Pi-limitation-inducible pho regulon, the products of which are involved in phosphate acquisition, may be anticipated. Indeed, the use of an uhpT-lacZ fusion revealed that much higher concentrations of the inducer glucose 6-P were required to elevate uhpT transcription when the pho regulon was expressed. This interference was the result of degradation of glucose 6-P by one of the products of the pho regulon, the periplasmic enzyme alkaline phosphatase. The specific form of interaction between the Pho and Uhp systems is designated inducer degradation.

An examination of the role of asp-177 in the His-Asp catalytic dyad of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: X-ray structure and pH dependence of kinetic parameters of the D177N mutant enzyme.

The role of Asp-177 in the His-Asp catalytic dyad of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides has been investigated by a structural and functional characterization of the D177N mutant enzyme. Its three-dimensional structure has been determined by X-ray cryocrystallography in the presence of NAD(+) and in the presence of glucose 6-phosphate plus NADPH. The structure of a glucose 6-phosphate complex of a mutant (Q365C) with normal enzyme activity has also been determined and substrate binding compared. To understand the effect of Asp-177 on the ionization properties of the catalytic base His-240, the pH dependence of kinetic parameters has been determined for the D177N mutant and compared to that of the wild-type enzyme. The structures give details of glucose 6-phosphate binding and show that replacement of the Asp-177 of the catalytic dyad with asparagine does not affect the overall structure of glucose 6-phosphate dehydrogenase. Additionally, the evidence suggests that the productive tautomer of His-240 in the D177N mutant enzyme is stabilized by a hydrogen bond with Asn-177; hence, the mutation does not affect tautomer stabilization. We conclude, therefore, that the absence of a negatively charged aspartate at 177 accounts for the decrease in catalytic activity at pH 7.8. Structural analysis suggests that the pH dependence of the kinetic parameters of D177N glucose 6-phosphate dehydrogenase results from an ionized water molecule replacing the missing negative charge of the mutated Asp-177 at high pH. Glucose 6-phosphate binding orders and orients His-178 in the D177N-glucose 6-phosphate-NADPH ternary complex and appears to be necessary to form this water-binding site.

Nonaggregating mutant of recombinant human hexokinase I exhibits wild-type kinetics and rod-like conformations in solution.

Hexokinase I governs the rate-limiting step of glycolysis in brain tissue, being inhibited by its product, glucose 6-phosphate, and allosterically relieved of product inhibition by phosphate. On the basis of small-angle X-ray scattering, the wild-type enzyme is a monomer in the presence of glucose and phosphate at protein concentrations up to 10 mg/mL, but in the presence of glucose 6-phosphate, is a dimer down to protein concentrations as low as 1 mg/mL. A mutant form of hexokinase I, specifically engineered by directed mutation to block dimerization, remains monomeric at high protein concentration under all conditions of ligation. This nondimerizing mutant exhibits wild-type activity, potent inhibition by glucose 6-phosphate, and phosphate reversal of product inhibition. Small-angle X-ray scattering data from the mutant hexokinase I in the presence of glucose/phosphate, glucose/glucose 6-phosphate, and glucose/ADP/Mg2+/AlF3 are consistent with a rodlike conformation for the monomer similar to that observed in crystal structures of the hexokinase I dimer. Hence, any mechanism for allosteric regulation of hexokinase I should maintain a global conformation of the polypeptide similar to that observed in crystallographic structures.

Heterogeneity of Glucose Transport in Rat Liver Microsomal Vesicles

Glucose transport across the membrane of rat liver microsomal vesicles was studied by a rapid filtration method in three different experimental systems: (i) inward transport in the presence of extravesicular glucose, (ii) efflux from passively preloaded vesicles, and (iii) efflux of glucose generated intravesicularly by glucose-6-phosphatase upon addition of glucose 6-phosphate were investigated. The apparent intravesicular glucose space estimated with the rapid filtration method was lower than the total microsomal glucose accessible space both the in the steady-state phase of uptake and at the starting point of efflux: 0.5 versus 2.3 μl/mg protein. The initial rate of influx/efflux was dependent on the extravesicular/intravesicular glucose concentration and was much lower than the rate of influx estimated previously by the light-scattering technique. Both influx and efflux could be inhibited byN-ethylmaleimide and possibly became saturable at high (>100 mM) glucose concentration. Known inhibitors of GLUT transporters (genistein, cytochalasin B, phloretin, and hexoses) did not affect glucose influx. The time course of glucose efflux from vesicles preincubated in the presence of glucose 6-phosphate was similar to that from glucose-loaded vesicles. These data together with that obtained previously (by a light-scattering technique; Marcolongo, P., Fulceri, R., Giunti, R., Burchell, A., and Benedetti, A. (1996)Biochem. Biophys. Res. Commun.219, 916–922) indicate that microsomal vesicles are heterogeneous regarding their glucose-transporting properties and that glucose transport is bidirectional and its feature meets the requirements of a facilitative transport.

Expression, Purification and Preliminary Crystallographic Studies of Human Hexokinase I

Abstract: Human hexokinase type I, one of the four isozymes consisting of a single polypeptide chain of about l 00 kDa, has been cloned in the pET expression plasmid in a truncated form lacking a segment of 11 apolar amino acids at the N-terminus. The protein has been overexpressed in E.coii and purified to homogeneity. Truncated hexokinase I has been crystallized in the presence of glucose 6-phosphate and of the ATP analogue AMP-PNP. The crystals belong to the monoclinic space group P21, with unit cell constants: a= 84.2 A, b = 177.7 A, c = 88.2 A, 13 = 90.8° and diffract to 3.0 A resolution.

Mannitol 1-phosphate mediates an inhibitory effect of mannitol on the activity and the translocation of glucokinase in isolated rat hepatocytes.

When tested in the presence of an inhibitor of sorbitol dehydrogenase, both mannitol and sorbitol caused a progressive inhibition of the detritiation of [2-3H]glucose in isolated rat hepatocytes. The purpose of the present work was to investigate the possibility that this effect was mediated by the regulatory protein of glucokinase. When added to hepatocytes, mannitol decreased the apparent affinity of glucokinase for glucose and increased the concentration of fructose required to stimulate detritiation, without affecting the concentration of fructose 1-phosphate. Its effect could be attributed to the formation of mannitol 1-phosphate, a potent agonist of the regulatory protein, which, similarly to fructose 6-phosphate, reinforces its inhibitory action. Formation of mannitol 1-phosphate in hepatocytes was dependent on the presence of mannitol and was stimulated by compounds that increase the concentration of glucose 6-phosphate. Liver extracts catalysed the conversion of mannitol to mannitol 1-phosphate about 7 times more rapidly in the presence of glucose 6-phosphate than of ATP. The glucose 6-phosphate-dependent formation was entirely accounted for by a microsomal enzyme, glucose-6-phosphatase and was not due to a loss of latency of this enzyme. In hepatocytes in primary culture, mannitol decreased the detritiation rate and counteracted the effect of fructose to stimulate glucokinase translocation. Taken together, these results strongly support a central role played by the regulatory protein in the control of glucokinase activity and translocation in the liver, as well as a feedback control exerted by fructose 6-phosphate on this enzyme.

Isoenzymatic pattern of glucose 6-phosphate dehydrogenase from Ascaris suum.

The isoenzymatic pattern of glucose 6-phosphate dehydrogenase (abbreviation G6PD) from Ascaris suum has been studied by vertical polyacrylamide gel (PAGE) and horizontal starch gel electrophoresis. After polyacrylamide gel electrophoresis, two stained zones could be identified. One corresponded to tetrazolium oxidase activity; since this zone was stained even in the absence of glucose 6-phosphate (G6P), non-specific staining could be detected. In the other zone of activity, seven regularly-spaced bands were identified by staining in the presence of G6P and NADP as substrates. By using starch gel electrophoresis, different electrophoretic patterns for G6PD have been observed in the muscular sac, intestine and reproductive system from A. suum. The existence of three different alleles of G6PD in the same individual suggests the existence of at least two genes for this enzyme.

Identification of the putative hexose-phosphate translocator of amyloplasts from cauliflower buds

Starch synthesis in amyloplasts isolated from cauliflower buds is strongly inhibited by the addition of micromolar concentrations of 4,4'-di-isothiocyano-2,2'-stilbenedisulphonic acid (DIDS). Using [3H]DIDS it was possible to label specifically a 31.6 kDa membrane protein of the envelope fraction of isolated amyloplasts. The intensity of the radioactive label was decreased in the presence of glucose 6-phosphate or dihydroxyacetone phosphate, indicating that this protein might be the amyloplastic hexosephosphate translocator.

Regulation of phospho enolpyruvate carboxylase from Crassula argentea: Effect of incubation with ligands and dilution on oligomeric state, activity, and allosteric properties

The relationship between the aggregation state and allosteric properties of purified phospho enolpyruvate carboxylase from Crassula argentea was examined using both kinetic and physical techniques. Analysis by native polyacrylamide gel electrophoresis showed that dilution induced a dissociation of the active tetramer to a less active dimer. Kinetic assays showed that inhibition of phospho enolpyruvate carboxylase by 5 m m malate measured at a saturating phospho enolpyruvate concentration rose to nearly 80% with increasing preassay dilution while the activity in the absence of malate remained constant. Kinetic bursts were observed when enzyme-initiated assays were measured at a subsaturating phospho enolpyruvate concentration. At saturating phospho enolpyruvate concentrations, however, increasing lags developed in response to increasing the preassay dilution of the enzyme. Further, dynamic laser-light scattering measurements showed that preincubation of the dilute enzyme with phospho enolpyruvate stabilized the tetramer while the presence of malate induced dimer formation. These observations confirm and extend earlier work with the extracted active malate insensitive night and less active, malate-sensitive day forms of the enzyme (Wu and Wedding [1985] Plant Physiol. 77, 667–675). Activity measured at subsaturating phospho enolpyruvate concentrations dropped with increasing preassay dilution of enzyme, while activation by 3.2 m m glucose 6-phosphate, assayed at a low phospho enolpyruvate concentration (0.044 m m), increased with dilution to nearly 400%. In this case activation results from a decrease in the control rate as the activity measured in the presence of glucose 6-phosphate was nearly constant, similar in effect to saturating phospho enolpyruvate in the assay. Glucose 6-phosphate induced tetramer formation of the dilute enzyme as measured by light-scattering similar to the effects induced by PEP. In addition, when diluted (dimeric) PEPC was preincubated with PEP or glucose 6-phosphate the enzyme became less sensitive to malate inhibition, while the active-site directed ligand 2-phosphoglycolate had no effect on malate inhibition. These results indicate that both the substrate PEP and the activator glucose 6-phosphate stabilize the active tetramer via binding and interaction at an activator site separate from the active site.

Purification and properties of sucrose-phosphate synthase from seeds of Pisum sativum

The aim of this work was to purify and characterize sucrose-phosphate synthase from 38 hr germinated seeds of Pisum sativum. Chromatography on ω-aminohexyl Sepharose 4B and PD 10 Sephadex G-25M followed by fast protein liquid chromatography on a Mono Q anion exchange column and a Superose 6 gel filtration column gave a preparation of specific activity 4.22 μmol min −1 mg −1 protein. SDS PAGE showed four major and five minor bands. Native molecular mass was 456 000. Activity was optimum at pH 7.0 and 5 mM magnesium chloride. Hyperbolic kinetics were found for UDPglucose, apparent K m 2.4 mM and, in the presence of glucose 6-phosphate, for fructose 6-phosphate. No inhibition was detected with either sucrose or sucrose phosphate. Glucose 6-phosphate and fructose-1,6-bisphosphate stimulated activity, but Pi and UDP were inhibitory.

An improved assay for hepatic glycogen synthase in liver extracts with emphasis on synthase R

An assay for measurement of optimal amounts of glycogen synthase R, the physiologically active form of the enzyme, in liver tissue extracts is described. Tissue extracts enriched in synthase R had a pH profile different from those reported for synthase D and synthase I. In tissue extracts, synthase I had a broad pH optimum but maximal activity was present at pH 7.0–9.0. Synthase D had a sharp pH optimum at pH 8.5 and had little activity at pH 7.0, either in the presence or in the absence of glucose 6-phosphate (G6P). In extracts enriched in synthase R, the pH optimum was 7.0–8.0 without G6P, but 8.0 with G6P. The synthase R activity without G6P rapidly decreased at a higher pH. The proportion of synthase in the physiologically active form traditionally has been reported as an activity ratio based upon the activity in the presence and absence of G6P. The assay has been performed at a single pH. Because of the differences in pH profile, we recommend that the enzyme be measured at pH 7.0 in the absence of G6P and pH 8.5–8.8 in the presence of G6P. In previous assays the substrate UDP-Glc concentration used often has been less than saturating, and the G6P concentration generally has been excessive. A substrate concentration of 11 m m UDP-Glc was found to be necessary for maximal activity. A G6P concentration of 2 m m is adequate for measurement of the D form of the enzyme.