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.     

Amiloride activation of hepatic microsomal glucose-6-phosphatase; activation of T1?

The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) in vitro by amiloride has been investigated in both intact and fully disrupted microsomes. The major effect of amiloride is a 4.5-fold reduction in the Km of glucose-6-phosphatase activity in intact diabetic rat liver microsomes. Amiloride also decreased the Km of glucose-6-phosphatase activity in intact liver microsomes isolated from starved rats 2.5-fold. Kinetic calculations, direct enzyme assays and direct transport assays all demonstrated that the site of amiloride action was T1, the hepatic microsomal glucose 6-phosphate transport protein. This is, to our knowledge, the first report of an activation of any of the proteins of the multimeric hepatic microsomal glucose-6-phosphatase complex.     

The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide on the rat liver microsomal glucose-6-phosphatase system

The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) on the reactions catalyzed by the glucose-6-phosphatase system of rat liver microsomes was studied. Modification of the intact microsomes by CMC leads to the inhibition of the glucose-6-phosphatase, pyrophosphate:glucose and carbamoyl-phosphate : glucose phosphotransferase activities of the system. The activities are restored by the disruption of the microsomal permeability barrier. The mannose-6-phosphate, pyrophosphate, and carbamoyl-phosphate phosphohydrolase activities of the intact as well as the disrupted microsomes were not affected by CMC. It follows from the results obtained that CMC inactivates the microsomal glucose-6-phosphate translocase, the inactivation is a result of the modification of a single sulfhydryl or amino group of the translocase; pyrophosphate, carbamoyl phosphate and inorganic phosphate are transported across the microsomal membrane without participation of the glucose-6-phosphate translocase; pyrophosphate and carbamoyl phosphate may act as the phosphate donors in the glucose phosphorylation reactions in vivo.     

Effect of insulin on the induction by dexamethasone of glucose-6-phosphohydrolase and translocase activities in cultured hepatoma cells

The ten-fold increase in glucose-6-phosphatase, previously reported, in 2S FAZA hepatoma cells exposed to dexamethasone, is completely blocked by low concentrations of insulin. At 3 x 10(-10) M insulin, the activity induced by 10(-6) M dexamethasone is reduced by half. The activity of intact microsomes, which reflects translocation of cytoplasmic glucose 6-phosphate into the endoplasmic reticulum, is induced by dexamethasone, but to a lesser extent than the hydrolase. Insulin also prevents this induction.     

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.     

Regulation of glucose 6-phosphatase in hepatic microsomes by thyroid and corticosteroid hormones

The regulation of glucose 6-phosphatase in hepatic microsomes by thyroid and corticosteroid hormones has been studied following the administration of 3,3',5-triiodo-L-thyronine and/or triamcinolone to hypophysectomized rats. The apparent Km for glucose-6-P in isolated ("intact") microsomes increased following administration of either hormone; there was little or no difference in the apparent Km when microsomes were treated with sodium deoxycholate ("disrupted"). In intact microsomes, triiodothyronine caused a 2.3-fold increase in the Vmax of glucose 6-phosphatase; triamcinolone, a 4-fold increase; and both hormones together, a 4.4-fold increase. Corresponding values for disrupted microsomes were: triiodothyronine, 3.7-fold; triamcinolone, 1.8-fold; both hormones, 3.3-fold. After triiodothyronine treatment, disruption of microsomes caused an over 5-fold increase in Vmax; after triamcinolone treatment, the increase was only 1.5-fold. This difference could not be explained by a change in the energy of activation of glucose 6-phosphatase in either intact or disrupted microsomes following hormone treatment. Glucose 6-phosphatase was localized by a cytochemical procedure; the reaction product was associated with 90% of the profiles in all microsomal preparations, except for those from triiodothyronine-treated rats, where less than 50% contained lead precipitate. Vesicles free of lead phosphate were isolated from sucrose gradients and accounted for less than 10% of the protein and glucose 6-phosphatase in all preparations, again except for those from triiodothyronine-treated rats, where they represented 40% of both the protein and glucose 6-phosphatase. The results are consistent with a model for glucose 6-phosphatase in which the substrate is transported across the microsomal membrane by a specific carrier before hydrolysis within the cisternae by a phosphohydrolase. It is suggested that the effect of triiodothyronine is mainly on the activity of the phosphohydrolase, and triamcinolone, on that of the carrier.     

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 K_m 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 see in many patients after administration of the drug pentamidine.     

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.     

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.     

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.     

The pentose phosphate pathway in the endoplasmic reticulum

Approximately the same levels of six of the seven enzymes catalyzing reactions of the pentose phosphate pathway are in the cisternae of washed microsomes from rat heart, spleen, lung, and brain. Renal and hepatic microsomes also have detectable levels of these enzymes except ribulose-5-phosphate epimerase and ribose-5-phosphate isomerase. Their location in the cisternae is indicated by their latencies, i.e. requirement for disruption of the membrane for activity. In addition, transketolase, transaldolase, and glucose-6-phosphatase, a known cisternal enzyme, are inactivated by chymotrypsin and subtilisin only in disrupted hepatic microsomes under conditions in which NADPH-cytochrome c reductase, an enzyme on the external surface, is inactivated equally in intact and disrupted microsomes. The failure to detect the epimerase and isomerase in hepatic microsomes is due to inhibition of their assays by ketopentose-5-phosphatase. Xylulose 5-phosphate is hydrolyzed faster than ribulose 5-phosphate. A mild heat treatment destroys hepatic xylulose-5-phosphatase and glucose-6-phosphatase without affecting acid phosphatase. These results plus the established wide distribution of glucose dehydrogenase, the microsomal glucose-6-phosphate dehydrogenase, and its localization to the lumen of the endoplasmic reticulum suggest that most mammalian cells have two sets of enzymes of the pentose phosphate pathway: one is cytoplasmic and the other is in the endoplasmic reticulum. The activity of the microsomal pentose phosphate pathway is estimated to be about 1.5% that of the cytoplasmic pathway.     

The effects of N-alkylmaleimides on the activity of rat liver glucose 6-phosphatase.

A series of N-alkylmaleimides has been synthesized and used to investigate the thiol groups that are essential for the activity of rat liver microsomal glucose 6-phosphatase. All of the N-alkylmaleimides inactivated glucose 6-phosphatase when preincubated with microsomes (microsomal fractions) at pH 6.5 and 30 degrees C. When enzyme activity was assayed in intact microsomes, the inactivation was non-linear with respect time, showing an initial rapid phase followed by a slower secondary phase. During the initial rapid phase the inactivation may apparently be completely reversed by disrupting the microsomal membrane with detergent. However, after longer exposure to N-alkylmaleimides the reversal is no longer complete. This observation was explained by the results obtained from studying the inactivation in detergent-disrupted microsomes. In this case glucose 6-phosphatase was also completely inactivated, but much more slowly than was seen in intact microsomes, and the process was linear with respect to time. When assayed in both intact and detergent-disrupted microsomes, glucose 6-phosphatase inactivation was dependent on the number of carbon atoms in the alkyl side chain of the N-alkylmaleimides; this dependence was much more marked in disrupted microsomes. Analysis of the data showed that in neither case was there a saturating effect at high concentrations of maleimide. The data have been interpreted to suggest that there are are least two thiol groups essential for activity located in two separate non-polar regions of the membrane-enzyme system. The conclusions are discussed in the light of the current model for the microsomal glucose 6-phosphatase system.     

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.     

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.     

Kinetic properties of the glucose 6-phosphatase of the liver from arthritic rats

According to previous reports, adjuvant-induced arthritic rats present reduced activities of the hepatic glucose 6-phosphatase. A kinetic study was done in order to characterize this phenomenon. Microsomes were isolated from livers of arthritic and control rats (Holtzman strain) and the glucose 6-phosphatase was measured at various temperatures (13-37 degrees C) and glucose 6-phosphate concentrations. Irrespective of the temperature, the enzyme from arthritic rats presented a reduction of both V(max) and K(M). Detergent treatment of liver microsomes from control rats increased the activity, but no increase was found when microsomes from arthritic rats were treated in the same way. The mannose 6-phosphatase activity of detergent-treated microsomes from arthritic rats was only 25% of the activity found with detergent-treated microsomes from control rats. Without detergent treatment, the mannose 6-phosphatase activities of both control and arthritic rats were minimal. The activation energy, derived from V(max), was not changed by arthritis. In vivo arthritic rats presented higher hepatic glucose 6-phosphate concentrations, a phenomenon that is consistent with a reduced activity of glucose 6-phosphatase. It was concluded that in arthritic rats, the hydrolase is probably reduced, without a similar change in the translocase activity.     

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.     

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.     

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.     

Changes in the glucose-6-phosphatase complex in hepatomas

Hepatomas tend to have a decreased glucose-6-phosphatase activity. We have observed phenotypic stability for this change in Morris hepatomas transplanted in rats. To determine if this decrease is selective for translocase functions or the hydrolase activity associated with glucose-6-phosphatase, we have compared activities in liver and hepatomas with glucose-6-phosphate or mannose-6-phosphate as substrates and with intact or histone-disrupted microsomes. In five out of seven subcutaneously transplanted rat hepatoma lines, the microsomal mannose-6-phosphatase activity was lower than in preparations from liver of normal or tumor-bearing rats. With liver microsomes and with most hepatoma microsomes, preincubation with calf thymus histones caused a greater increase in mannose-6-phosphatase than in glucose-6-phosphatase activity. In studies with liver and hepatoma microsomes there were similar increases in mannose-6-phosphatase activity with total calf thymus histones and arginine-rich histones. A smaller increase was seen with lysine-rich histones. The effect of polylysine was similar to the action of lysine-rich histones. There was only a small effect with protamine at the same concentration (1 mg/ml). Rat liver or hepatoma H1 histones gave only about half the activation seen with core nucleosomal histones. Our data suggested that microsomes of rat hepatomas tend to have decreased translocase and hydrolase functions of glucose-6-phosphatase relative to activities in untransformed liver. (Mol Cell Biochem122: 17–24, 1993)     

On the nature of the interaction between 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid and microsomal glucose-6-phosphatase. Evidence for the involvement of sulfhydryl groups of the phosphohydrolase

The effect of 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (DIDS) on microsomal glucose 6-phosphate hydrolysis has been reinvestigated and characterized in order to elucidate the topological and functional properties of the interacting sites of the glucose-6-phosphatase. The studies were performed on microsomal membranes, partially purified and reconstituted glucose-6-phosphatase preparations and show the following. (a) DIDS inhibits activity of the glucose-6-phosphatase of native microsomes as well as the partially purified glucose-6-phosphatase. (b) Inhibition is reversed when the microsomes and the partially purified phosphohydrolase, incorporated into asolectin liposomes, are modified with Triton X-114. (c) Treatment of native microsomes with DIDS and the following purification of glucose-6-phosphatase from these labeled membranes leads to an enzyme preparation which is labeled and inhibited by DIDS. (d) Preincubation of native microsomes or partially purified glucose-6-phosphatase with a 3000-fold excess of glucose 6-phosphate cannot prevent the DIDS-induced inhibition. (e) Inhibition of glucose-6-phosphatase by DIDS is completely prevented when reactive sulfhydryl groups of the phosphohydrolase are blocked by p-mecuribenzoate. (f) Reactivation of enzyme activity is obtained when DIDS-labeled microsomes are incubated with 2-mercaptoethanol or dithiothreitol. Therefore, we conclude that inhibition of microsomal glucose 6-phosphate hydrolysis by DIDS cannot result from binding of this agent to a putative glucose-6-phosphate-carrier protein. Our results rather suggest that inhibition is caused by chemical modification of sulfhydryl groups of the integral phosphohydrolase accessible to DIDS attack itself. An easy interpretation of these results can be obtained on the basis of a modified conformational model representing the glucose-6-phosphatase as an integral channel-protein located within the hydrophobic interior of the microsomal membrane [Schulze et al. (1986) J. Biol. Chem. 261, 16,571-16,578].