Radiation inactivation analysis of rat liver microsomal glucose-6-phosphatase

Radiation inactivation analysis was utilized to estimate the sizes of the units catalyzing the various activities of hepatic microsomal glucose-6-phosphatase. This technique revealed that the target molecular weights for mannose-6-P phosphohydrolase, glucose-6-P phosphohydrolase, and carbamyl-P:glucose phosphotransferase activities were all about Mr 75,000. These results are consistent with the widely held view that all of these activities are catalyzed by the same protein or proteins. Certain observations indicate that the molecular organization of microsomal glucose-6-phosphatase is better described by the conformational hypothesis which envisions the enzyme as a single covalent structure rather than by the substrate transport model which requires the participation of several physically separate polypeptides. These include the findings: 1) that the target sizes for glucose-6-P phosphohydrolase and carbamyl-P:glucose phosphotransferase activities were not larger than that for mannose-6-P phosphohydrolase in intact microsomes and 2) that the target size for glucose-6-P phosphohydrolase in disrupted microsomes was not less than that observed in intact microsomes. These findings are most consistent with a model for glucose-6-phosphatase of a single polypeptide or a disulfide-linked dimer which spans the endoplasmic reticulum with the various activities of this multifunctional enzyme residing in distinct protein domains.     

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.     

Microsomal membrane permeability and the hepatic glucose-6-phosphatase system. Interactions of the system with D-mannose 6-phosphate and D-mannose.

We have proposed that glucose-6-phosphatase (EC 3.1.3.9) is a two-component system consisting of (a) a glucose-6-P-specific transporter which mediates the movement of the hexose phosphate from the cytosol to the lumen of the endoplasmic reticulum (or cisternae of the isolated microsomal vesicle), and (b) a nonspecific phosphohydrolase-phosphotransferase localized on the luminal surface of the membrane (Arion, W.J., Wallin, B.K., Lange, A.J., and Ballas, L.M. (1975) Mol. Cell. Biochem. 6, 75-83). Additional support for this model has been obtained by studying the interactions of D-mannose-6-P and D-mannose with the enzyme of untreated (i.e. intact) and taurocholate-disrupted microsomes. An exact correspondence was shown between the mannose-6-P phosphohydrolase activity at low substrate concentrations and the permeability of the microsomal membrane to EDTA. The state of intactness of the membrane influenced the kinetics of mannose inhibition of glucose-6-P hydrolysis; uncompetitive and noncompetitive inhibitions were observed for intact and disrupted microsomes, respectively. The apparent Km for glucose-6-P was smaller with intact preparations at mannose concentrations above 0.3 M. Mannose significantly inhibited total glucose-6-P utilization by intact microsomes, whereas D-glucose had a stimulatory effect. Both hexoses markedly enhanced the rate of glucose-6-P utilization by disrupted microsomes. The actions of mannose on the glucose-6-phosphatase of intact microsomes fully support the postulated transport model. They are predictable consequences of the synthesis and accumulation of mannose-6-P in the cisternae of microsomal vesicles which possess a nonspecific, multifunctional enzyme on the inner surface and a limiting membrane permeable to D-glucose, D-mannose, glucose-6-P, but impermeable to mannose-6-P. The latency of the mannose-6-P phosphohydrolase activity is proposed as a reliable, quantitative index of microsomal membrane integrity. The inherent limitations of the use of EDTA permeability for this purpose are discussed.     

Thermal stability of microsomal glucose-6-phosphatase

The thermal stability of glucose-6-phosphatase in rat liver microsomes was examined in untreated and cholate-treated microsomes. Activity of the enzyme was measured with both glucose-6-P and mannose-6-P as substrates. Heat treatment did not cause glucose-6-phosphatase activity to decline to zero with a single rate constant in untreated microsomes. Instead, heat treatment produced an enzyme with a small residual activity that was stable. The residual level of activity was not stimulated by addition of detergent. In untreated microsomes the energies of activation for the processes of decay were different for glucose-6-phosphatase and mannose-6-phosphatase activities, suggesting that the rate-limiting steps for the hydrolysis of these compounds were different. Treatment of microsomes with detergent increased the rate constants for the thermal decay of glucose-6-phosphatase by about 150 times, and, in contrast to untreated microsomes, glucose-6-phosphatase and mannose-6-phosphatase decayed to zero with a single rate constant in cholate-treated microsomes. Also, rate constants for thermal inactivation of glucose-6-phosphatase and mannose-6-phosphatase were the same in cholate-treated microsomes. Removal of cholate increased the stability of glucose-6-phosphatase but did not regenerate the form of the enzyme present in untreated microsomes. The data for the stability of glucose-6-phosphatase under different conditions provide evidence that the enzyme can exist in at least five different stable states that are enzymatically active.     

The importance of membrane integrity in kinetic characterizations of the microsomal glucose-6-phosphatase system

The transport model of glucose-6-phosphatase (EC 3.1.3.9) was recently challenged by a report that detergent treatment had no effect on the presteady state kinetics of glucose-6-P hydrolysis catalyzed at 0 degree C by the enzyme in liver microsomes previously frozen in 0.25 M mannitol (Zakim, D., and Edmondson, D. E. (1982) J. Biol. Chem. 257, 1145-1148). The lack of response to detergent is shown to be the expected consequence of the conditions used in the presteady state measurements. First, when the assay temperature was reduced from 30 to 0 degree C the depression in the glucose-6-P phosphohydrolase activity of intact microsomes (i.e. the system) was much greater than that of fully disrupted microsomes (i.e. enzyme). This indicates that temperature influences transport much more than hydrolysis of glucose-6-P. As a result, the contribution of a small fraction of enzyme associated with disrupted structures is markedly exaggerated, so it becomes the predominant hydrolytic activity before detergent treatment. Second, freezing microsomes in 0.25 M mannitol caused such extensive disruption that all of the activity manifest at 0 degree C could be attributed to enzyme in disrupted structures. The present findings underscore the importance of assessing the state of intactness of "untreated" microsomes and quantifying the contribution of the disrupted component in kinetic analyses of the glucose-6-phosphatase system. The proposition that the detergent-induced changes in the kinetic properties of glucose 6-phosphatase represent removal of constraints imposed on the enzyme by the membrane environment rather than increased access of enzyme to substrate is critically analyzed.     

The role of the membrane in the regulation of activity of microsomal glucose-6-phosphatase

The factors regulating glucose-6-phosphatase (EC 3.1.3.9) activity and substrate specificity in hepatic microsomes were studied by determining the rate-limiting reaction for the hydrolysis of glucose-6-P, and by examining the effect of detergent activation on phosphotransferase activity. Examination of the pre-steady state kinetics of glucose-6-phosphatase revealed that the steady state rate is determined by the rate of hydrolysis of the enzyme-P intermediate. Treatment of the enzyme with detergent does not alter the extent of the rapid release of glucose per mg of protein, but activates the steady state rate of catalytic turnover. Specificity of the enzyme was evaluated by comparing the effects of mannose and glucose as phosphate acceptors in the phosphotransferase reaction catalyzed by glucose-6-phosphatase. Untreated glucose-6-phosphatase discriminates against mannose as compared with glucose in that mannose and glucose bind to the enzyme-P intermediate of untreated enzyme, but mannose is not an acceptor of Pi. Mannose is an acceptor, however, after treatment of microsomes with detergent. These data cannot be explained in terms of the currently accepted "compartmentation" model for the regulation of glucose-6-phosphatase. The detergent-induced changes in kinetic properties appear to reflect alterations in the intrinsic characteristics of glucose-6-phosphatase, which could result from interaction with its membrane environment.     

The kinetics of intact microsome glucose-6-phosphatase are sigmoid at physiologic glucose 6-phosphate concentrations

The kinetics of rat liver glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) were studied with intact and detergent-disrupted microsomes from normal and diabetic rats. Glucose-6-P concentrations employed (12 microM to 1.0 mM) spanned the physiologic range. With the enzyme of intact microsomes from both groups, plots of v versus [glucose-6-P] were sigmoid. Hanes plots (i.e. [glucose-6-P]/v versus [glucose-6-P]) were biphasic (concave upwards). A Hill coefficient of 1.45 was determined with substrate concentrations between 12 and 133 microM. Disruption of microsomal integrity abolished these departures from classic kinetic behavior, indicating that sigmoidicity may result from cooperative interaction of glucose-6-P with the glucose-6-phosphatase system at the substrate translocase specific for glucose-6-P. With the enzyme from normal rats the [glucose-6-P] at which the enzyme was maximally sensitive to variations in [glucose-6-P] (which we term "Smax"), determined from plots of dv/d [glucose-6-P] versus [glucose-6-P], was in the physiologic range. The Smax of 0.13 mM corresponded well with the normal steady-state hepatic [glucose-6-P] of 0.16 mM, consistent with glucose-6-phosphatase's function as a regulatory enzyme. With the diabetic enzyme, in contrast, values were 0.30 and 0.07 mM for the Smax and steady-state level, respectively. We suggest that the decreasing sensitivity of glucose-6-phosphatase activity to progressively diminishing glucose-6-P concentration, inherent in its sigmoid kinetics, constitutes a mechanism for the preservation of a residual pool of glucose-6-P for other hepatic metabolic functions in the presence of elevated concentrations of glucose-6-phosphatase such as in diabetes.     

The characteristics of liver glucose-6-phosphatase in the envelope of isolated nuclei and microsomes are identical

We have compared the characteristics of glucose-6-phosphatase (EC 3.1.3.9) in the envelope of purified nuclei and microsomes from rat liver. The latency of mannose-6-P hydrolysis, permeability to EDTA, and susceptibility of the enzyme to protease-mediated inactivation all indicated that the permeability barrier defined by the envelope in situ is significantly disrupted in isolated nuclei (i.e. in vitro). Latency of mannose-6-P hydrolysis was demonstrated to provide a quantitative measure of the degree of nuclear membrane disruption. Electron micrographs confirmed the existence of substantial regions of the envelope in vitro where the permeability barrier to EDTA was intact (i.e. an "intact component"). The kinetics of glucose-6-phosphatase catalyzed by the intact component was obtained by subtracting the contribution of enzyme in disrupted regions from the total enzymic activity of untreated nuclei. The characteristics of glucose-6-phosphatase in intact and fully disrupted membranes of nuclei were indistinguishable from microsomes with respect to (a) the kinetics of glucose-6-P hydrolysis, (b) the effects of incubations with mannose-6-P, N-ethylmaleimide, and protease from Bacillus amyloliquefaciens, (c) the extremely high latency of carbamyl phosphate:glucose phosphotransferase activity, and (d) both the patterns of response of activity and the change in latency of glucose-6-phosphatase induced by fasting, experimental diabetes, and cortisol injection. Our results show clearly that apparent differences in the glucose-6-phosphatase activity of untreated preparations of nuclei and microsomes are simply expressions of significant differences in the degree of intactness of their respective permeability barriers. Since flattened cisternae, characteristic of the rough endoplasmic reticulum in situ, are preserved in intact regions of the envelope of isolated nuclei, the present findings constitute the most direct and definitive evidence to date that the properties of glucose-6-phosphatase in the endoplasmic reticulum in situ are faithfully reproduced with intact microsomes.     

Evidence for glucose-6-phosphate transport in rat liver microsomes

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.     

Anomer specificity of glucose-6-phosphatase and glucokinase

The anomeric form of glucose produced by glucose-6-phosphatase was studied using an apparatus that specifically measures beta-D-glucose. The time course of beta-D-glucose formation from glucose-6-P by glucose-6-phosphatase is essentially linear. In the presence of mutarotase, this rate is reduced to 70% of that obtained in the absence of mutarotase. When detergent treated microsomes were used, the rate of beta-D-glucose formation is unaffected by mutarotase. These results suggest that only beta-anomer of glucose is produced by microsomal glucose-6-phosphatase and this specificity is determined by translocase for glucose-6-P or glucose. It was also demonstrated that alpha-D-glucose is the substrate for glucokinase.     

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.     

Membrane effects on hepatic microsomal glucose-6-phosphatase.

1) Rat liver microsomes exhibit only a weak hydrolyzing activity towards galactose 6-phosphate. Disruption of the microsomal vesicles does not change the apparent Michaelis constant for this substrate but enhances the apparent maximum velocity. 2) The inhibition of microsomal glucose-6-phosphatase (EC 3.1.3.9) by galactose 6-phosphate is of the competitive type in intact and disrupted microsomal vesicles, suggesting that both substrates are hydrolyzed by the same enzyme. 3) The high degree of latency found for the hydrolysis of galactose 6-phosphate compared to glucose 6-phosphate indicates the presence of a carrier for glucose 6-phosphate in the microsomal membrane. 4) Since glucose as a product is not trapped inside the microsomal vesicles, this sugar probably is able to penetrate the microsomal membrane.     

The glucose-6-phosphatase system in rat hepatic microsomes displays hyperbolic kinetics at physiological glucose 6-phosphate concentrations

The kinetics of rat liver glucose-6-phosphatase (EC 3.1.3.9) were studied in intact and detergent-disrupted microsomes from normal and diabetic rats at pH 7.0 using two buffer systems (50 mM Tris-cacodylate and 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and glucose-6-P varied from 20 microM to 10 mM. Identical data were obtained when the phosphohydrolase activity was quantified by a colorimetric determination of Pi or by measuring 32Pi formed during incubations with [32P]glucose-6-P. In every instance the initial rate data displayed excellent concordance with that expected for a reaction obeying Michaelis-Menten kinetics. The present findings agree with recently reported results of Traxinger and Nordlie (Traxinger, R. R., and Nordlie, R. C. 1987) J. Biol. Chem. 262, 10015-10019) that glucose-6-phosphatase activity in intact microsomes exhibits hyperbolic kinetics at concentrations of glucose-6-P above 133 microM, but fail to confirm their finding of sigmoid kinetics at substrate concentrations below 133 microM. We conclude that glucose-6-P hydrolysis conforms to a hyperbolic function at concentrations of glucose-6-P existing in livers of normal and diabetic rats in vivo.     

Evidence for the participation of independent translocation for phosphate and glucose 6-phosphate in the microsomal glucose-6-phosphatase system. Interactions of the system with orthophosphate, inorganic pyrophosphate, and carbamyl phosphate

The interactions of Pi, PPi, and carbamyl-P with the hepatic glucose-6-phosphatase system were studied in intact and detergent-disrupted microsomes. Penetration of PPi and carbamyl-P into intact microsomes was evidenced by their reactions with the enzyme located exclusively on the luminal surface. Lack of effects of carbonyl cyanide m-chlorophenylhydrazone and valinomycin + KCl indicated that pH gradients and/or membrane potentials that could influence the kinetics of the system are not generated during metabolism of PPi and glucose-6-P by intact microsomes. With disrupted microsomes, only competitive interactions were seen among glucose-6-P, Pi, PPi, and carbamyl-P. With intact microsomes, Pi, PPi, and carbamyl-P were relatively weak, noncompetitive inhibitors of glucose-6-phosphatase, and PPi hydrolysis was inhibited competitively by Pi and carbamyl-P but noncompetitively by glucose-6-P. Analysis of the kinetic data in combination with findings from other studies that a variety of inhibitors of the glucose-6-P translocase (T1) does not affect PPi hydrolysis provide compelling evidence that permeability of microsomes to Pi, PPi, and carbamyl-P is mediated by a second translocase (T2). Some properties of the microsomal anion transporters are described. If the characteristics of the glucose-6-phosphatase system as presently defined in intact microsomes apply in vivo, glucose-6-P hydrolysis appears to be the predominant, if not the exclusive, physiologic function of the system. Both the "noncompetitive character" and the relative ineffectiveness of Pi as an inhibitor of glucose-6-phosphatase of intact microsomes result from the rate limitation imposed by T1 that prevents equilibration of glucose-6-P across the membrane. In microsomes from fed rats, where T1 is less rate restricting, about one-half as much Pi was required to give 50% inhibition compared with microsomes from fasted or diabetic rats. Thus, any treatment or agent that alters the kinetic relationship between transport and hydrolysis of glucose-6-P (e.g. endocrine or nutritional status) is an essential consideration in analyses of kinetic data for the glucose-6-phosphatase system.     

Calcium sequestration activity in rat liver microsomes. Evidence for a cooperation of calcium transport with glucose-6-phosphatase

Mechanisms regulating the energy-dependent calcium sequestering activity of liver microsomes were studied. The possibility for a physiologic mechanism capable of entrapping the transported Ca2+ was investigated. It was found that the addition of glucose 6-phosphate to the incubation system for MgATP-dependent microsomal calcium transport results in a marked stimulation of Ca2+ uptake. The uptake at 30 min is about 50% of that obtained with oxalate when the incubation is carried out at pH 6.8, which is the pH optimum for oxalate-stimulated calcium uptake. However, at physiological pH values (7.2-7.4), the glucose 6-phosphate-stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. The Vmax of the glucose 6-phosphate-stimulated transport is 22.3 nmol of calcium/mg protein per min. The apparent Km for calcium calculated from total calcium concentrations is 31.9 microM. After the incubation of the system for MgATP-dependent microsomal calcium transport in the presence of glucose 6-phosphate, inorganic phosphorus and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. In the system for the glucose 6-phosphate-stimulated calcium uptake, glucose 6-phosphate is actively hydrolyzed by the glucose-6-phosphatase activity of liver microsomes. The latter activity is not influenced by concomitant calcium uptake. Calcium uptake is maximal when the concentration of glucose 6-phosphate in the system is 1-3 mM, which is much lower than that necessary to saturate glucose-6-phosphatase. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose-6-phosphatase multicomponent system. The physiological implications of such a cooperation are discussed.     

Liver glucose-6-phosphatase activity is modulated by physiological intracellular Ca2+ concentrations

The effect of varying concentrations of free Ca2+ on the formation of Pi from mannose-6-P or of Pi and [U-14C]glucose from [U-14C]glucose-6-P was investigated in isolated fasted rat hepatocytes made permeable by freezing and in liver microsomes. Free Ca2+ concentration was adjusted by the use of Ca-EGTA buffers. In permeabilized cells, glucose-6-phosphatase (EC 3.1.3.9) activity was inhibited up to 50% and in intact microsomes up to 70% by increasing free Ca2+ concentrations from 0.01 to 10 microM. The inhibition was reversible and competitive with respect to glucose-6-P. Treatment of microsomes with 0.4% deoxycholate exposed 90% of latent mannose-6-phosphatase activity which was insensitive to Ca2+. The results indicate that Ca2+ affects the glucose-6-P translocase rather than the phosphohydrolase component. It is concluded that the glucose-6-phosphatase system is modulated by changes in Ca2+ concentrations in the range of those occurring in the liver cell upon hormonal stimulation.     

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.     

The mechanism of histone activation of the hepatic microsomal glucose-6-phosphatase system: a novel method to assay glucose-6-phosphatase activity

The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) by histone 2A has been investigated in both intact and disrupted microsomes. Histone 2A increased the Vmax and decreased the Km of glucose-6-phosphatase in intact microsomes but had no effect on glucose-6-phosphatase activity in disrupted microsomes. Histone 2A was shown to activate glucose-6-phosphatase in intact microsomes by disrupting the membrane vesicles and thereby allowing the direct measurement of the activity of the latent glucose-6-phosphatase enzyme. The study demonstrated that disrupting microsomes with histone 2A is an excellent method for directly assaying glucose-6-phosphatase activity as it poses none of the problems encountered with all of the previously used methods.     

Pentamidine activates T1 the hepatic microsomal glucose 6-phosphate transport protein of the glucose-6-phosphatase system.

The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) in vitro by pentamidine has been investigated in both intact and fully disrupted microsomes. The major effect of pentamidine is a 4.7-fold reduction in the Km of glucose-6-phosphatase activity in intact diabetic rat liver microsomes. The site of action of pentamidine is T1 the hepatic microsomal glucose 6-phosphate transport protein. The activation of T1 by pentamidine may contribute to the disturbed blood glucose homeostasis seen in many patients after the administration of the drug pentamidine.     

Interaction of mannose-6-phosphate with the hysteretic transition in glucose-6-phosphate hydrolysis in intact liver microsomes.

We showed previously that glucose-6-phosphatase activity was characterised in intact liver microsomes by a hysteretic transition between a rapid and a slower catalytic form of the enzyme. We have now further investigated the substrate specificity of these two kinetic forms. It was found that the pre-incubation of intact microsomes with mannose-6-phosphate or glucose-6-phosphate (50 microM for 30 s) suppressed the burst in glucose-6-phosphatase activity, that the hysteretic transition was reversible and that mannose-6-phosphate inhibited glucose-6-phosphate hydrolysis during the first seconds of incubation, but not anymore after the burst. Our results indicate (i) that mannose-6-phosphate is recognised by the enzyme and can promote the hysteretic transition and (ii) that the transient phase is part of the catalytic mechanism itself.