Mutations in the glucose-6-phosphatase gene are associated with glycogen storage disease types 1a and 1aSP but not 1b and 1c.

Glycogen storage disease (GSD) type 1, which is caused by the deficiency of glucose-6-phosphatase (G6Pase), is an autosomal recessive disease with heterogenous symptoms. Two models of G6Pase catalysis have been proposed to explain the observed heterogeneities. The translocase-catalytic unit model proposes that five GSD type 1 subgroups exist which correspond to defects in the G6Pase catalytic unit (1a), a stabilizing protein (1aSP), the glucose-6-P (1b), phosphate/pyrophosphate (1c), and glucose (1d) translocases. Conversely, the conformation-substrate-transport model suggests that G6Pase is a single multifunctional membrane channel protein possessing both catalytic and substrate (or product) transport activities. We have recently demonstrated that mutations in the G6Pase catalytic unit cause GSD type 1a. To elucidate whether mutations in the G6Pase gene are responsible for other GSD type 1 subgroups, we characterized the G6Pase gene of GSD type 1b, 1c, and 1aSP patients. Our results show that the G6Pase gene of GSD type 1b and 1c patients is normal, consistent with the translocase-catalytic unit model of G6Pase catalysis. However, a mutation in exon 2 that converts an Arg at codon 83 to a Cys (R83C) was identified in both G6Pase alleles of the type 1aSP patient. The R83C mutation was also demonstrated in one homozygous and five heterogenous GSD type 1a patients, indicating that type 1aSP is a misclassification of GSD type 1a. We have also analyzed the G6Pase gene of seven additional type 1a patients and uncovered two new mutations that cause GSD type 1a.

N-acetylglucosamine-insensitive glucose phosphorylation in isolated hepatocytes from rats fasted for 48 h.

Glucose phosphorylation in isolated hepatocytes was studied by the release of 3H from D-[2-3H]glucose. Glucokinase activity was decreased by fasting rats for 48 h and was further reduced in cells by adding 30 mM GlcNAc, a potent competitive inhibitor. Although this treatment resulted in the loss of more than 97% of glucokinase activity in hepatocytes, glucose phosphorylation proceeded at an appreciable rate. These observations demonstrate the involvement of a high -K0.5 enzyme system in addition to glucokinase in hepatocyte glucose phosphorylation.

Vanadate: a potent inhibitor of multifunctional glucose-6-phosphatase.

Vanadate has been found to be a potent inhibitor of both the hydrolytic and synthetic activities of the multifunctional enzyme glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9). The enzyme, when studied in both microsomal preparations and in situ using permeable isolated hepatocytes, is inhibited by micromolar concentrations of vanadate. The inhibition by vanadate is greater in detergent-treated than in untreated microsomes. In both the microsomal preparations and permeable hepatocytes, the inhibition by vanadate is competitive with the phosphate substrate and is greater for the phosphotransferase than the hydrolase activity of the enzyme. The Ki values of vanadate for carbamyl-phosphate : glucose phosphotransferase and glucose-6-phosphate phosphohydrolase determined with permeable hepatocytes are in good agreement with the values determined with detergent-dispersed microsomes. The previously described inhibition of glucose-6-phosphate phosphohydrolase by ATP (Nordlie, R.C., Hanson, T.L., Johns, P.T. and Lygre, D.G. (1968) Proc. Natl. Acad. Sci. USA 60, 590-597) can now be explained by the vanadium contamination of the commercially available ATP samples used. In contrast with glucose-6-phosphatase, hepatic glucokinase and hexokinase were not inhibited by vanadate. Physiological implications and utilitarian experimental applicability of vanadate as a selective metabolic probe, based on these observations, are suggested.

Mammalian carbamyl phosphate : glucose phosphotransferase and glucose-6-phosphate phosphohydrolase: extended tissue distribution.

Carbamyl phosphate : glucose phosphotransferase and glucose-6-phosphate (Glc-6-P) phosphohydrolase activities have beeh demonstrated in pancreas, adrenals, brain, testes, spleen, and lung. Catalysis of these activities by classical multifunctional glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) has been firmly established for the first four of these tissues on the basis of characteristic catalytic properties of the transferase pH-activity profiles, apparent Km values for carbamyl phosphate and glucose, substrate specificity, susceptibility to inhibition by molybdate, and activation by deoxycholate. Additional such activity due to non-specific acid (and alkaline) phosphatase action also is indicated at very high glucose concentrations. The possible physiological significance of the newly-elucidated presence of glucose-6-phosphatase-phosphotransferase in these various tissues, in addition to previously extensively studied liver, kidney, and mucosa of small intestine, is discussed briefly.

Comparative reactivity of carbamyl phosphate and glucose 6-phosphate with the glucose-6-phosphatase of intact microsomes.

The ability of glucose 6-phosphate and carbamyl phosphate to serve as substrates for glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) of intact and disrupted microsomes from rat liver was compared at pH 7.0. Results support carbamyl phosphate and glucose 6-phosphate as effective substrates with both. Km values for carbamyl phosphate and glucose 6-phosphate were greater with intact than with disrupted microsomes, but Vmax values were higher with the latter. The substrate translocase-catalytic unit concept of glucose-6-phosphatase function is thus confirmed. The Km values for 3-O-methyl-D-glucose and D-glucose were larger when determined with intact than with disrupted microsomes. This observation is consistent with the involvement of a translocase specific for hexose substrate as a rate-influencing determinant in phosphotransferase activity of glucose-6-phosphatase.

Hysteresis at near-physiologic substrate concentrations underlies apparent sigmoid kinetics of the glucose-6-phosphatase system.

Although Canfield and Arion (J. Biol. Chem. 263, 7458-7460 (1990)) have described the kinetics as hyperbolic, Traxinger and Nordlie (J. Biol. Chem. 262, 10015-10019 (1987)) reported sigmoid kinetics in the glucose-6-phosphatase system of intact microsomes at near-physiologic glucose-6-P concentrations. We show here that apparent sigmoidal kinetics, most clearly seen as sharp upward inflections in Hanes plots as substrate concentration approaches zero, are a consequence of the hysteretic lag in product formation during the first minutes of incubation of the enzyme with low concentrations of substrate. The appearance of sigmoidicity, observed when reaction velocities are calculated from changes in Pi concentration between 0 and 6 min of incubation, is not present when velocity is determined from slopes of [product]-time plots after linearity is achieved. The Km,glucose-6-P value, 0.86 mM, based on these hysteresis-corrected velocity values determined with intact microsomes from normal, control rats at low substrate concentrations, approached the upper limit of physiologic hepatic glucose-6-P concentrations. This suggests that glucose-6-phosphatase activity may be regulated by factors other than substrate concentrations alone. We propose that the hysteretic behavior, not sigmoid kinetics of the glucose-6-phosphatase enzyme system, may be a prime regulatory feature.

Inhibition of the glucose-6-phosphatase system by N-bromoacetylethanolamine phosphate, a potential affinity label for auxiliary proteins.

N-Bromoacetylethanolamine phosphate (BAEP) has been used previously as an affinity label to study the hexose phosphate binding sites of fructose-6-P, 2-kinase:fructose-2, 6-bisphosphatase (Sakakibara et al. (1984) J. Biol. Chem. 259, 14023-14028). We have employed this compound to probe components of the glucose-6-phosphatase system using a combination of time-dependent and immediate inhibition kinetic techniques. Inhibition of D-glucose-6-phosphate (G6P) phosphohydrolase activity of native microsomes was irreversible and time- and inhibitor-concentration-dependent. Only a partial time-dependent, irreversible inhibition of the PPi phosphohydrolase activity of native microsomes was observed. BAEP inhibited PPi:glucose phosphotransferase activity of native microsomes in a concentration-dependent, irreversible manner which was more extensive than that seen with PPi phosphohydrolase, but less extensive than was observed with G6P phosphohydrolase. Disruption of microsomal integrity by detergent-treatment either prior to incubation with BAEP or subsequent to preliminary incubation with BAEP but prior to assay for activity abolished the time-dependent inhibition. These irreversible, time- and concentration-dependent inhibitory actions of BAEP thus are manifest at a site or sites where the intact membrane-bound enzyme first makes contact with substrates G6P and PPi. An additional site of inhibition by BAEP, through relatively weak, reversible competitive inhibition at the active catalytic site, is indicated by classical steady-state kinetic analysis. The irreversible, time- and concentration-dependent inhibitions by BAEP seen with G6P and PPi as substrates strongly suggest the potential utility of radio-labeled BAEP as an affinity label for the identification and ultimate isolation and study of uncharacterized auxiliary components of the glucose-6-phosphatase system.

Responses of nuclear glucose-6-phosphatase to diabetes and to hydrocortisone administered to normal and diabetic rats differ from those of the microsomal enzyme.

The effect of alloxan-diabetes, and of pharmacological doses of hydrocortisone administered to normal and diabetic rats, on carbamyl phosphate:glucose phosphotransferase and D-glucose-6-phosphate phosphohydrolase (EC 3.1.3.9) activities of isolated hepatic nuclei and microsomes were studied by assay at pH 7 in the absence and presence of deoxycholate. Hormonally related alterations both in activity levels and in the activation by the detergent (i.e. latency) of activities of the two cellular structural elements differed significantly. Most strikingly, (a) a 3--4-fold increase in the levels of activities of nuclei was seen in response either to diabetes or to hydrocortisone administered to normal rats whether or not detergent was added to preparations prior to assay; (b) the normally low degree of stimulation by detergent of activities of nuclei was unaltered in diabetes, and (c) administration of the glucocorticoid to diabetic rats decreased activity levels and increased their activation by detergent. Directly contrasting responses were noted with isolated microsomal preparations. Fundamental differences in the enzymes in these two organelle preparations are thus demonstrated. It appears that both synthetic and hydrolytic activities of this enzyme of nuclei may be manifest in the presence of requisite substrates, and that activities of this organelle may become increasingly prominent under certain hormonally perturbed conditions.

Stimulation by polyamines of carbamylphosphate:glucose phosphotransferase and glucose-6-phosphate phosphohydrolase activities of multifunctional glucose-6-phosphatase.

The effects of added polyamines on carbamylphosphate (carbamyl-P):glucose phosphotransferase and glucose-6-phosphate (Glc-6-P) phosphohydrolase activities of rat hepatic D-Glc-6-P phosphohydrolase (EC 3.1.3.9) of intact and detergent-treated microsomes have been investigated. With the former preparation, in the presence of 1.4 mM phosphate substrate and 90 mM D-glucose (phosphotransferase), 1 mM spermine, spermidine, and putrescine activated Glc-6-P phosphohydrolase 67%, 57%, and 35%, respectively. Carbamyl-P:glucose phosphotransferase, under comparable conditions, was activated 57%, 34%, and 18%. NH+4 (0.25--5.0 mM) produced at best but a minor activation (0--14%), while poly(L-lysine) (Mr = 3400; degree of polymerization 16) equimolar relative to other polyamines with respect to ionized free amino groups activated the hydrolase 358% and the transferase 222%. Treatment of microsomes with the detergent deoxycholate reduced, but did not abolish, polyamine-induced activation. The stimulatory effects of polyamines persisted in the presence of excess catalase, indicating their independence from H2O2 formation; and were eliminated in the presence of Ca2+. Kinetic analysis revealed that all tested polyamines decreased the apparent Michaelis constant values for carbamyl-P and Glc-6-P, but had no effect on the Km for glucose. Poly(L-lysine) increased the V value for both Glc-6-P phosphohydrolase and apparent V values for phosphotransferase extrapolated to infinite concentrations of either carbamyl-P or glucose. The other tested polyamines elevated only this last velocity parameter. It is proposed that a major mechanism by which polyamines activate glucose-6-phosphatase-phosphotransferase is through their electrostatic interactions with phospholipids of the membrane of the endoplasmic reticulum of which this enzyme is a part. Conformational alterations thus induced may in turn affect catalytic behavior. It is suggested that polyamines, or similar positively charged peptides, might participate in the cellular regulation of synthetic and hydrolytic activities of glucose-6-phosphatase.

Time-dependent inhibition of glucose 6-phosphatase by 3-mercaptopicolinic acid.

3-Mercaptopicolinate (3-MP) inhibits D-glucose-6-phosphate (G6P) phosphohydrolase activity of the glucose-6-phosphatase system (Bode et al. (1993) Biochem. Cell Biol. 71, 113-121). We therefore attempted to maximize the inhibition by varying the physical state of microsomes, the concentration of 3-MP, and the time of preliminary incubation of 3-MP with the enzyme. The inhibition was irreversible and time- and inhibitor-concentration-dependent, with G6P phosphohydrolase activity of intact rat liver microsomes, but there was no inhibition with detergent-treated microsomes. The effectiveness of 3-MP as a time-dependent inhibitor of glucose 6-phosphatase was demonstrated in situ by measuring glycogenolysis in isolated, perfused livers from fed rats. We first exposed the livers to 2 mM 3-MP for 40 min, and then assessed the inhibitory effects on glycogenolysis. It was lowered by 50%. These observations establish that 3-MP at the mM level may be useful as an experimental probe in the study of the role(s) of G6P in the regulation of glycogenolysis as well as glycogenesis. Further, they validate the use of much lower (microM) concentrations of 3-MP to block gluconeogenesis (at the phosphoenolpyruvate carboxykinase step) without interfering with glucose 6-phosphatase. We also explored the mechanism of 3-MP inhibition. The time-dependent inhibition of carbamoyl-phosphate:glucose phosphotransferase activity with microsomes incubated with 1 mM 3-MP for 60 or 90 min and then assayed with 1 mM carbamoyl phosphate and 180 mM glucose was modest compared with inhibition of G6P phosphohydrolase. When G6P production by carbamoyl-phosphate:glucose phosphotransferase was reduced by decreasing glucose concentration to 60 mM, no inhibition by 3-MP was discernible. There was no inhibition of inorganic pyrophosphatase activity. These studies support the model of time-dependent, irreversible reaction of 3-MP with the G6P translocase component of the glucose-6-phosphatase system.

Stimulation by 3-mercaptopicolinate of net glucose uptake by perfused livers from diabetic rats.

Glucose uptake/production was studied as a function of varied glucose loads in isolated perfused livers from glucagon-treated alloxan-diabetic rats. Uptake of D-[U-14C]glucose was seen at all levels studied - 9.5-71 mM. In studies with unlabelled D-glucose carried out in the absence of 3-mercaptopicolinate, livers of diabetic rats showed a net production of glucose with perfusate glucose levels less than 22 mM. Above this level, these livers exhibited a time- and concentration-dependent net uptake of glucose for a period of 20-30 min. When 4 mM 3-mercaptopicolinate, which inhibited gluconeogenesis from endogenous substrates, was included in perfusates, a continuous net uptake of unlabelled glucose was observed at all levels above 4 mM. This lowering of the null-point, cross-over glucose concentration was shown to relate mechanistically to the observed reduction in steady-state hepatic glucose 6-phosphate level produced by mercaptopicolinate. The need for supplemental mechanisms of glucose utilization by high Km hepatic enzyme(s) operative in the virtual absence of insulin-dependent glucokinase also is indicated by these observations and by kinetic analysis.

Hysteretic behavior of the hepatic microsomal glucose-6-phosphatase system.

Carbamyl-P:glucose and PPi:glucose phosphotransferase, but not inorganic pyrophosphatase, activities of the hepatic microsomal glucose-6-phosphatase system demonstrate a time-dependent lag in product production with 1 mM phosphate substrate. Glucose-6-P phosphohydrolase shows a similar behavior with [glucose-6-P] less than or equal to 0.10 mM, but inorganic pyrophosphatase activity does not even at the 0.05 or 0.02 mM level. The hysteretic behavior is abolished when the structural integrity of the microsomes is destroyed by detergent treatment. Calculations indicate that an intramicrosomal glucose-6-P concentration of between 20 and 40 microM must be achieved, whether in response to exogenously added glucose-6-P or via intramicrosomal synthesis by carbamyl-P:glucose or PPi:glucose phosphotransferase activity, before the maximally active form of the enzyme system is achieved. It is suggested that translocase T1, the transport component of the glucose-6-phosphatase system specific for glucose-6-P, is the target for activation by these critical intramicrosomal concentrations of glucose-6-P.

The progressive effects of fasting on glucose phosphorylation by isolated rat hepatocytes: the involvement of a high K0.5 enzyme.

The effects of varied durations of food deprivation on the rates and kinetics of glucose phosphorylation by isolated rat hepatocytes have been examined. Glucokinase activity was measured concurrently in extracts from these cells prepared from livers of rats which had fasted for 0, 24, 48 and 72 h. Significant levels of hepatocyte glucose phosphorylation were noted even when glucokinase levels were extrapolated to zero. The K0.5-glucose value of 33 mM in cells from fed rats increased to 48 mM after a 72-h fast. It is concluded that a high K0.5 glucose-phosphorylating enzyme or enzymes compensatory to insulin-dependent glucokinase is/are involved in rat liver glucose phosphorylation.

Metabolic intermediates as potential regulators of glucose-6-phosphatase.

Twenty-five metabolites of glucose, gluconeogenic substrates, and related compounds were examined as potential inhibitors of glucose-6-phosphatase (EC 3.1.3.9) catalytic unit and substrate transport function, using disrupted and intact rat liver microsomes. Inhibitions (competitive) were noted with six. Calculated per cent inhibitions with presumed near-physiologic concentrations of inhibitor and substrate were small. However, when hepatic fructose-1-P concentration is elevated in response to a fructose load, inhibition of glucose-6-phosphatase by fructose-1-P may play a regulatory role, along with fructose-1-P-associated deinhibition of glucokinase, by directing glucose-6-P away from glucose formation and towards glycogen synthesis and glycolysis.

Glucose-6-phosphatase Activity in Copper-Deficient Rats.

Copper deficiency has been reported to cause glucose intolerance in rats by interfering with normal glucose utilization. Accordingly, copper deficiency was produced in rats to study its effects on glucose-6-P phosphohydrolase and carbamyl-P: glucose phosphotransferase activities of hepatic glucose-6-phosphatase (EC 3.1.3.9), a major enzyme involved in maintaining glucose homeostasis. When measured in homogenates treated with deoxycholate, total glucose-6-P phosphohydrolase was 23% lower and total carbamyl-P:glucose phosphotransferase was 17% lower in copper-deficient rats compared to controls. Latency, or that portion of total activity that is not manifest unless the intact membranous components are disrupted with deoxycholate also was lower in copper-deficient rats. Glucose-6-P phosphohydrolase was 5% latent in copper-deficient rats compared to 24% in controls and carbamyl-P : glucose phosphotransferase was 55% latent in copper-deficient rats compared to 65% in controls. The decrease in latency appears to compensate for the lower total enzyme activities in such a manner as to allow the net expression of these activities in the intact membranous components of the homogenate to remain unaltered by copper deficiency. It thus appears unlikely that copper deficiency affects glucose homeostasis in vivo by altering the net rate of glucose-6-P hydrolysis or synthesis by glucose-6-phosphatase. These observations are interpreted on the basis of a multicomponent glucose-6-phosphatase system in which the total enzyme activity expressed in intact membranous preparation is limited by substrate specific translocases that transport substrate to the membrane-bound catalytic unit. A decrease in latency can then be interpreted as a functional increase in translocase activity and may constitute a compensating mechanism for maintaining constant glucose homeostasis when glucose-6-phosphatase catalytic activity is depressed as it is in copper deficiency.