Glucose 6-phosphate Phosphohydrolase Research Articles

The hepatic glucose-6-phosphatase system in Ehrlich-ascites-tumour-bearing mice

To examine the effects of the presence of Ehrlich ascites tumours on both the catalytic unit and the substrate/product translocase components of the glucose-6-phosphatase system in vivo, we isolated microsomes from the livers of control and tumour-bearing mice. Samples were analysed immunochemically for the quantity of catalytic unit, stabilizing protein and translocases T2 and T3 proteins. In comparison experiments, a variety of kinetic studies were performed. The most striking findings in tumour-bearing mice were: a 2.5-fold increase in the quantity of translocase T2 protein; increases in the Km and Vmax. for glucose 6-phosphate phosphohydrolase; and a decrease in the Km value for carbamoyl phosphate (carbamoyl-P) of carbamoyl-P:glucose phosphotransferase, all with intact microsomes. The percentage latency at Vmax. decreased for PPi phosphohydrolase and for glucose 6-phosphate phosphohydrolase, but was unaffected for carbamoyl-P:glucose phosphotransferase. These observations support a tumour-related increase in translocase T2 capacity in vivo, as it transports Pi from the microsomal lumen to the medium and carbamoyl-P or PPi from the medium to the microsomal lumen.

Studies on a pyrophosphatase and glucose-6-phosphatase from Aspergillus oryzae

An inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) isolated from Aspergillus oryzae is associated with a glucose-6-phosphatase (glucose 6-phosphate phosphohydrolase, EC 3.1.3.9). It is also associated with a pyrophosphate-glucose phosphotransferase. These enzyme activities are heat stable, have acidic pH optima, and require no metallic ions for activity. Studies, including the determination of kinetic parameters, indicate that these catalytic activities are due to a single enzyme. This enzyme has a mol. wt of about 60 800 as determined by gel filtration. The enzyme also has low levels of acid phosphatase activity. A. oryzae possesses a second inorganic pyrophosphatase which has a low pH optimum and could be separated from the other catalytic activities.

Kinetics of the Glucose 6-Phosphate-Glucose Exchange Activity and Glucose Inhibition of Glucose 6-Phosphatase of Intact and Disrupted Rat Liver Microsomes

1. The steady state kinetics of the glucose 6-phosphate-glucose exchange catalyzed by glucose 6-phosphatase was determined for intact and disrupted rat liver microsomes. An assay for the exchange activity was devised that entailed measurements of the influences of added glucose on the rates of formation of inorganic phosphate and [14C]glucose from [14C]glucose 6-phosphate. Values obtained with this assay were in excellent agreement with those obtained by measurements of the rates of incorporation of [14C]glucose into glucose 6-phosphate. Activity-pH profiles for the exchange activity were markedly similar to those for the glucose 6-phosphate phosphohydrolase activity. In contrast to other glucose phosphotransferase activities, the exchange activity of untreated microsomes was manifest over a broad range of pH, including neutrality, and the pH optimum (pH 6) was unaffected by disrupting the microsomal structure. With both intact and disrupted microsomes the maximal rates of exchange were greater than the maximal rates of glucose 6-phosphate hydrolysis indicating that the hydrolysis of the phosphoryl-enzyme is the slow step in the phosphohydrolase reaction. 2. Lineweaver-Burk plots describing glucose inhibition of glucose 6-phosphate hydrolysis of intact structures were unlike any classical type of inhibition. As the concentration of glucose was raised the maximal rate of hydrolysis and the apparent Michaelis constant were decreased and the slopes of the lines increased slightly (i.e. the family of plots intersected at a point within the third quadrant). In contrast, the double reciprocal plots describing glucose inhibition of the enzyme of disrupted microsomes intersected at a common point within the second quadrant. The coordinates of the intersection point defined the kinetic constants for the exchange activity. Conversely, primary plots of data for the exchange activity of disrupted microsomes intersected at points within the third quadrant. The coordinates of these intersection points defined the kinetic constants for the hydrolase activity. The value of the inhibitor constant for glucose was greatest with intact microsomes. This could not be attributed to a lesser sensitivity or a decreased accessibility of the enzyme of intact structures to the hexose. 3. The results of this and other recent studies were analyzed in the light of the proposal by Lueck, Herrman and Nordlie ((1972) Biochemistry 11, 2792–2799) that the differences in catalytic properties of the enzyme from untreated and disrupted microsomes can be attributed to detergent- or pH-induced modifications of the individual rate constants associated with the terminal steps of the phosphohydrolase and phosphotransferase activities. This proposal was found to be untenable because it fails to account for (a) the observed changes in the Michaelis constant for glucose 6-phosphate in the phosphohydrolase reaction and (b) the quantitative differences in the responses to detergent supplementation of various phosphotransferase activities of the enzyme.

Evaluation of the Rate-determining Steps and the Relative Magnitude of the Individual Rate Constants for the Hydrolytic and Synthetic Activities of the Catalytic Component of Liver Microsomal Glucose 6-Phosphatase

Abstract 1. Steady state kinetic constants were determined at pH 6 for various phosphohydrolase and phosphotransferase activities catalyzed by glucose 6-phosphatase of liver microsomes that were supplemented with 0.4% sodium taurocholate and assayed in the presence of albumin. Km and Vmax values were evaluated for reactions with glucose 6-phosphate, carbamyl phosphate, inorganic pyrophosphate, mannose 6-phosphate, ATP, and phosphoenolpyruvate as phosphoryl donor compounds and d-glucose and d-mannose as phosphoryl acceptor compounds. 2. The order of decreasing reactivity of these substrates was carbamyl phosphate g inorganic pyrophosphate g mannose 6-phosphate = glucose 6-phosphate >> ATP g phosphoenolpyruvate. Phosphotransferase activities with d-mannose were significantly faster than the corresponding glucose phosphotransferase activities. With the exception of phosphoenolpyruvate, values for Vmax and Km for a given phosphate substrate were greater in the phosphotransferase reactions. The Km for a given substrate in the phosphotransferase reaction varied with the specific nature and concentration of the second substrate. Values of Km for the phosphoryl donor substrates were greatest with mannose as the acceptor, while Km values for the phosphoryl acceptor varied directly with the reactivity of the phosphoryl donor compound. 3. These observations were shown to be incompatible with the conclusion from an earlier study that the step in the mechanism was the formation of the phosphoryl enzyme from the binary complex of enzyme and phosphoryl donor compound. Rather, it is concluded that the hydrolysis of phosphoryl enzyme is the slowest step in the hydrolysis of all substrates that possess reactivity equal to or greater than glucose 6-phosphate. Moreover, no individual rate constant could be considered as the rate-limiting factor in these reactions. The earlier conclusions appear to be valid at pH 6 in the cases of phosphoenolpyruvate, ATP, or other nucleotides where additional steric or electrostatic factors restrict the rate of formation of phosphoryl enzyme. 4. An analysis of the data for all activities permitted the assignment of upper and lower limits on the relative magnitude of the individual rate constants for the glucose 6-phosphate phosphohydrolase activity and means by which these limits may be refined are discussed.

The requirement for membrane integrity in the inhibition of hepatic glucose 6-phosphatase by sulfhydryl reagents and taurocholate

Exposures of intact rat liver microsomes to various sulfhydryl reagents or to concentrations of taurocholate below 1 mM inhibited glucose 6-phosphate phosphohydrolase activity. The inhibitions were abolished by subsequent disruption of membrane integrity by higher concentrations of taurocholate or 1M NH 4OH. Also the reagents were ineffective when tested directly with disrupted microsomes. Thus, a requirement for an intact membrane structure in the action of these reagents is indicated. Moreover, the involvement of a second component of the endoplasmic reticulum in the catalysis of glucose 6-phosphate hydrolysis is suggested.

The Nature of pH Discriminant Differences in the Behavior of Various Phosphoanhydrides, Mixed Phosphate Anhydrides, and Phosphate Esters as Substrates and Inhibitors with Microsomal Glucose 6-Phosphatase

Abstract The relative abilities of a variety of phosphoanhydrides, phosphate esters, and mixed phosphate anhydrides (acyl phosphate compounds) to function (a) as substrates for hydrolase and phosphotransferase activities of rat liver microsomal d-glucose 6-phosphate phosphohydrolase (EC 3.1.3.9) and (b) as inhibitors of glucose 6-phosphate phosphohydrolase activity have been considered as a function of reaction pH. Competitive inhibitions of glucose 6-phosphate phosphohydrolase activity by adenosine 5'-triphosphate, inorganic pyrophosphate, mannose 6-phosphate, and carbamyl phosphate have been observed over the entire range of hydrogen ion concentrations studied, pH 5 to 8. In contrast, hydrolytic and synthetic activities with pyrophosphate and adenosine 5'-triphosphate have been observed only at neutral and acid pH, while glucose 6-phosphate, mannose 6-phosphate, carbamyl phosphate (and several other acyl phosphate anhydrides) are effective substrates at alkaline as well as lower pH values. Under conditions where both parameters may be determined, Km values for various compounds functioning as substrates for phosphohydrolase and phosphotransferase activities agree well with Ki values for these compounds acting as competitive inhibitors. Activity-pH profiles for both hydrolase and phosphotransferase (with glucose as acceptor molecule) activities are, with each substrate, essentially identical. It is concluded from these observations (a) that all these compounds may bind to a common enzymic active site at all pH values to produce binary enzyme-phosphoryl compound complexes, as indicated by inhibition data; (b) that assay pH does not discriminate between hydrolase and phosphotransferase activity of the enzyme, per se, since both types of activity are demonstrable at alkaline, as well as lower pH values with certain tested compounds; and (c) that pH discriminant effects (reactivity, or inhibition at high compared with lower pH values, or both) thus are dependent upon the nature of the phosphate substrate rather than the type of reaction involved. It is concluded, from these observations and a consideration of relevant earlier studies, that discrimination by assay pH with respect to the modes of reactivity of various phosphate compounds (ability to function as substrates and inhibitors as contrast with ability to function only as inhibitors) is manifest at an intermediate step in the over-all reaction mechanism, that involving the dissociation of binary enzyme-phosphoryl compound complexes to produce a phosphorylenzyme intermediate. The possible physiological implications of these observations are discussed briefly in terms of roles and regulation at pH 7 of activities of glucose 6-phosphatase, which may be under rigid metabolite control.

The Inhibition by Physiological Orthophosphate Concentrations of Hydrolytic and Synthetic Activities of Liver Glucose 6-Phosphatase

Abstract The inhibition by orthophosphate of glucose 6-phosphate phosphohydrolase and inorganic pyrophosphate-glucose phosphotransferase activities of rat liver glucose 6-phosphatase (EC 3.1.3.9) has been studied in detail. Significant inhibitions of both activities were noted over the entire range of pH values studied—pH 5.0 to 7.5 (hydrolase) or pH 5.0 to 6.5 (phosphotransferase). The detergents cetyltrimethylammonium bromide (Cetrimide) and lysolecithin significantly lowered Ki values for Pi with both activities. For example, at pH 7.5, a Ki value of 18 mm was noted in the absence of detergents, while preliminary treatment of microsomes with Cetrimide, to 0.05% (w/v), reduced this value to 2.0 mm, and inclusion of 0.4 mm lysolecithin in assay mixtures led to a decrease in Ki to 4.6 mm. Because of differences in the extents of accompanying modifying effects of detergents on Km values for substrates, net inhibition by Pi of glucose 6-phosphate phosphohydrolase activity was considerably more extensively potentiated by detergents than was that of phosphotransferase activity. Both in the absence and presence of detergents, inhibitions were kinetically competitive with respect to glucose 6-phosphate and pyrophosphate and non-competitive with respect to glucose in the phosphotransferase reaction. The mutually competitive nature of interactions of orthophosphate with adenosine triphosphate, bicarbonate, and pyrophosphate noted in inhibition studies carried out with phosphohydrolase activity also was shown. A physiologically significant regulatory role for Pi, through its effects on activities of this enzyme, is suggested on the basis of these experimental observations.

Inhibitory Effect of Physiological Bicarbonate Ion Levels on the Activities of Glucose 6-Phosphate Phosphohydrolase

Abstract Rat liver microsomal glucose 6-phosphate phosphohydrolase has been shown to possess two further catalytic activities: the hydrolysis of inorganic pyrophosphate to orthophosphate and a pyrophosphate-glucose phosphotransferase activity. Physiological concentrations of bicarbonate ion (10 to 50 mm) have been found to inhibit all three activities. A detailed kinetic analysis of the nature and extent of this inhibition has been carried out, with both fresh and deoxycholate-treated microsomes. The effect of pH on the degree of inhibition, at constant bicarbonate ion levels, suggests that bicarbonate ion rather than carbon dioxide is the inhibitory species. The inhibition by bicarbonate ion has been found to be competitive with respect to phosphate substrate and noncompetitive with respect to glucose. Measurements at varied substrate levels, when plotted according to the method of Dixon, show that 1/v is a linear function of [I], suggesting that bicarbonate ion is binding at the catalytic active site. The Ki values calculated for the three activities (≅20 mm) show close agreement, providing further support for the belief that a common active site, and possibly a common enzyme-phosphoryl intermediate, is involved in all three activities. It is suggested that the observed inhibition of glucose 6-phosphate phosphohydrolase by bicarbonate ion may explain the observation of Hastings and Longmore that increased bicarbonate-carbon dioxide levels result in increased glycogen accumulation in rat liver slices.

Biological Regulation of Liver Microsomal Inorganic Pyrophosphate-Glucose Phosphotransferase, Glucose 6-Phosphatase, and Inorganic Pyrophosphatase: Differential Effects of Fasting on Synthetic and Hydrolytic Activities

The effects of fasting on hydrolytic and synthetic activities of liver microsomal glucose 6-phosphate phosphohydrolase (EC 3.1.3.9) have been investigated. Differential responses of the two types of activity were observed. Glucose 6-phosphate and inorganic pyrophosphate phosphohydrolase activity levels were elevated after a 48-hour fast, while inorganic pyrophosphate-glucose phosphotransferase activity responded relatively slightly, based on assays carried out in the absence of detergent. However, when homogenates were treated with deoxycholate prior to assay, parallel increases in all three activities were obtained. This latter observation constitutes additional substantiation for the multifunctional nature of microsomal glucose 6-phosphatase. The differential response patterns, and modifying effects of detergent thereon, were observed regardless of the hydrogen ion concentration in assay mixtures in the pH range 4.5 to 7. Kinetic analyses revealed that the differential response patterns were not due simply to fasting-induced changes in Km values, for the noted differences persisted when activity levels were expressed on the basis of Vmax values extrapolated to infinite concentrations of all substrates. Phosphotransferase activity of enzyme newly synthesized or newly activated in response to fasting appears to be blocked at the level of phosphoryl transfer from phosphoryl-enzyme intermediate to glucose. The observed differential patterns of response are consistent with the established hydrolytic role for the enzyme in fasted animals, and contrast with previously observed modes of response of these activities to glucocorticoid administration and experimental diabetes.

Differential Effects of Palmityl Coenzyme A on Liver Microsomal Inorganic Pyrophosphate-Glucose Phosphotransferase and Glucose 6-Phosphate Phosphohydrolase

Abstract The effects of palmityl coenzyme A on both hydrolytic and inorganic pyrophosphate-glucose phosphotransferase activities of glucose 6-phosphate phosphohydrolase (EC 3.1.3.9) of rat liver microsomes have been investigated. It was found that the acyl coenzyme A compound may either stimulate or inhibit, in a pH- and concentration-dependent, activity-discriminating manner. Marked activation of the phosphotransferase activity (to a maximum of 350% of control levels in one instance) was observed under a wide variety of conditions, while the glucose 6-phosphate phosphohydrolase activity was at best but modestly elevated (to a maximum of 120% of control levels) and was significantly inhibited by palmityl coenzyme A under most conditions tested. A concomitant extensive activation of phosphotransferase and inhibition of phosphohydrolase activity was observed with certain levels of the acyl coenzyme A compound. Effective concentrations of palmityl coenzyme A coincided with established levels of hepatic long chain fatty acyl coenzyme A derivatives. Coenzyme A, palmitate, and acetyl coenzyme A were without effect on enzymic activities. Maximal phosphotransferase activity was observed at pH 4.3 with untreated microsomal preparations. This maximum was shifted to pH 6 when assays were carried out in the presence of 4.1 x 10-5 m palmityl coenzyme A, and a significant amount of activity was observed at pH 7.0 under these conditions. It is suggested that changes, in vivo, of functional levels of the hydrolytic and synthetic activities of this enzyme in animals in various altered hormonal states, for example, diabetes, possibly may be mediated in part through these activity-discriminating effects of long chain acyl coenzyme A compounds.

Biological Regulation of Inorganic Pyrophosphate-Glucose Phosphotransferase and Glucose 6-Phosphatase: ACTIVATION BY TRIAMCINOLONE, IN VIVO, IN THE PRESENCE OF ACTINOMYCIN D

Abstract Previous studies (4) on the effects of glucocorticoid therapy on liver microsomal glucose 6-phosphate phosphohydrolase (EC 3.1.3.9) and associated inorganic pyrophosphate-glucose phosphotransferase activity have been extended to include investigations of the effects, in vivo, of the synthetic steroid triamcinolone when administered to animals in which protein biosynthesis had been blocked by treatment with actinomycin D. Actinomycin (10 µg/100 g of body weight) was administered daily for 3 days by intraperitoneal injection to a group of 11 rats. Triamcinolone (1.0 mg/100 g of body weight) was injected daily ½ hour later into five of these animals. Enzymic activities were assayed in fresh liver homogenates which were supplemented either with 0.1 part of 2% sodium deoxycholate solution or with an equal volume of distilled water. Glucose 6-phosphate phosphohydrolase and pyrophosphate-glucose phosphotransferase activity levels increased, respectively, to 139% and 177% of comparable values for activities in animals receiving actinomycin only, based on assays carried out in the absence of detergent. However, statistically significant differences in corresponding activity values for the two groups of animals disappeared when assays were carried out with detergent-supplemented homogenates. These findings constitute additional evidence supporting our earlier suggestion (4) that the principal effect of glucocorticoid therapy on the various activities of microsomal glucose 6-phosphatase is that of activation of preexisting enzyme; furthermore, they indicate that this process is independent of protein biosynthesis.

The Inhibition by Phlorizin of Kidney Microsomal Inorganic Pyrophosphate-Glucose Phosphotransferase and Glucose 6-Phosphatase

Abstract Glucose 6-phosphatase from rat liver and kidney microsomes previously has been shown to catalyze an inorganic pyrophosphate-glucose phosphotransferase reaction. Nordlie and Soodsma recently have suggested that formation and then degradation of glucose 6-phosphate by the combined action of phosphotransferase and phosphohydrolase activities of this enzyme may under certain conditions constitute part of a mechanism for facilitated transport of glucose across the kidney tubular cell. To attempt to gain additional insight regarding the possible significance of such a physiological role for the enzyme, studies of the effect of phlorizin, a potent inhibitor of tubular sugar transport, on both phosphohydrolase and phosphotransferase activities of this enzyme were undertaken. Inhibition, in vitro, of both glucose 6-phosphate phosphohydrolase and pyrophosphate-glucose phosphotransferase activities of kidney homogenates and microsomal suspensions prepared in isotonic sucrose solution was noted over the entire range of phlorizin concentrations studied, 6.7 x 10-5 m to 6.7 mm. Sensitivity of activities to inhibition by this compound was either partially or completely abolished by supplementation of the homogenates or microsomal suspensions, to 0.2% (w/v), with sodium deoxycholate, sodium cholate, digitonin, Triton X-100, or Tween 20. Phlorizin was without discernible effect on activity of partially purified enzyme preparations obtained by fractional ammonium sulfate precipitation of deoxycholate-dispersed microsomal suspensions. Inhibition of phosphotransferase activity in homogenates or microsomes was markedly potentiated by the cationic detergent trimethylhexadecylammonium bromide. Kinetic analyses indicated that inhibition of both phosphohydrolase and phosphotransferase activities by phlorizin was of the classical noncompetitive type. A value of approximately 0.7 mm for Ki for phlorizin was determined in the absence of detergent; the value was reduced to 0.2 InM when trimethylhexadecylammonium bromide was added (to 0.2%, w/v) to enzyme preparations prior to assay for activity. The effects of the administration in vivo of phlorizin on phosphotransferase activity of the enzyme were also studied. Reductions in activity per mg of kidney protein of as much as 18% were produced by intravenous injection of 0.1-g doses of phlorizin to rats. The data obtained appear to rationalize the initially apparently contradictory reports of earlier workers regarding the effects (or lack of effect) of phlorizin on glucose 6-phosphatase. The experimental observations reported in this paper also are interpreted to constitute added support for, but not direct proof of, the possible involvement of synthetic and hydrolytic activities of this enzyme in kidney glucose transport. It is suggested that the modifying effects of various detergents on phlorizin-sensitivity of the enzyme are manifest through selective action on both active and inactive silent phlorizin-binding sites in the enzyme preparation.