Topographical localization and characterization of microsomal glucose-6-phosphatase binding sites accessible to 4,4'-diazidostilbene 2,2'-disulfonic acid

The effect of the photoactivated reagent 4,4'-diazidostilbene 2,2'-disulfonic acid (DASS) on rat liver microsomal glucose-6-phosphatase has been investigated in order to analyze the accessibility and the chemical nature of functional sites of the integral enzyme protein. The following results were obtained. (i) When native rat liver microsomes are irradiated with the photoactive reagent, the activity of glucose-6-phosphatase is progressively inhibited. However, complete reactivation is obtained by modification of the DASS-labeled microsomes with Triton X-114. (ii) Inhibition of glucose-6-phosphatase is also reversed when the DASS-labeled microsomes are treated with p-mercuribenzoate or dithiothreitol. (iii) When native microsomes are labeled with DASS an intensely fluorescent adduct is formed whose emission and excitation maximum corresponds with those obtained when cysteine or 3-mercaptopropionic acid are irradiated in the presence of the photolabile reagent. (iv) The data from fluorescence measurements show that p-mercuribenzoate and dithiothreitol reduce fluorescence labeling of the microsomes whereas Triton modification of the DASS-labeled membranes does not affect the DASS-induced fluorescence. (v) Glucose 6-phosphate hydrolysis of the partially purified glucose-6-phosphatase is also inhibited as observed with native microsomes. The DASS-induced inhibition is reversed and prevented by p-mercuribenzoate; however, the partially purified enzyme cannot be reactivated by Triton X-114. (vi) When glucose-6-phosphatase is partially purified from the DASS-labeled microsomes this enzyme preparation is fluorescence labeled and inhibited. From these results we conclude that DASS directly reacts with the integral phosphohydrolase mainly by chemical modification of essential sulfhydryl groups of the enzyme protein accessible from the cytoplasmic surface of the native microsomal membrane. The Triton-induced reactivation of the glucose-6-phosphatase of DASS-labeled microsomes is explained in terms of conformational changes of the integral protein elicited during modification of the surrounding membrane by detergent.     

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].     

Modulation of the activity of hepatic glucose-6-phosphatase by methylthioadenosine sulfoxide.

Methylthioadenosine sulfoxide (MTAS), an oxidized derivative of the cell toxic metabolite methylthioadenosine has been used in elucidating the relevance of an interrelationship between the catalytic behavior and the conformational state of hepatic glucose-6-phosphatase and in characterizing the transmembrane orientation of the integral unit in the microsomal membrane. The following results were obtained: (1) Glucose 6-phosphate hydrolysis at 37 degrees C is progressively inhibited when native microsomes are treated with MTAS at 37 degrees C. In contrast, glucose 6-phosphate hydrolysis of the same MTAS-treated microsomes assayed at 0 degrees C is not inhibited. (2) Subsequent modification of the MTAS-treated microsomes with Triton X-114 reveals that glucose-6-phosphatase assayed at 37 degrees C as well as at 0 degrees C is inhibited. (3) Although excess reagent is separated by centrifugation and the MTAS-treated microsomes diluted with buffer before being modified with Triton the temperature-dependent effect of MTAS on microsomal glucose-6-phosphatase is not reversed at all. (4) In native microsomes MTAS is shown to inhibit glucose-6-phosphatase noncompetitively. The subsequent Triton-modification of the MTAS-treated microsomes, however, generates an uncompetitive type of inhibition. (5) Preincubation of native microsomes with MTAS completely prevents the inhibitory effect of 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS) as well as 4,4'-diazidostilbene 2,2'-disulfonate (DASS) on glucose-6-phosphatase. (6) Low molecular weight thiols and tocopherol protect the microsomal glucose-6-phosphatase against MTAS-induced inhibition. (7) Glucose-6-phosphatase solubilized and partially purified from rat liver microsomes is also affected by MTAS in demonstrating the same temperature-dependent behavior as the enzyme of MTAS-treated and Triton-modified microsomes. From these results we conclude that MTAS modulates the enzyme catalytic properties of hepatic glucose-6-phosphatase by covalent modification of reactive groups of the integral protein accessible from the cytoplasmic surface of the microsomal membrane. The temperature-dependent kinetic behavior of MTAS-modulated glucose-6-phosphatase is interpreted by the existence of distinct catalytically active enzyme conformation forms. Detergent-induced modification of the adjacent hydrophobic microenvironment additionally generates alterations of the conformational state leading to changes of the kinetic characteristics of the integral enzyme.     

Studies on the interactions between phospholipids and membrane-bound enzymes in microsomes. Effects of phospholipases C on the glucose-6-phosphatase system of rat liver microsomes

The role of phospholipids in the glucose-6-phosphatase system, including glucose-6-P phosphohydrolase and glucose-6-P translocase, was studied in rat liver microsomes by using phospholipases C and detergents. In the time course experiments on detergent exposure, the maximal activation of glucose-6-P phosphohydrolase varied according to the nature of the detergent used. On treatment of microsomes with phospholipase C of C. perfringens, the activity of glucose-6-P phosphohydrolase without detergent (i.e. without rupture of translocase activity) was gradually decreased with the progressive hydrolysis of phosphatidylcholine and phosphatidylethanolamine on the microsomal membrane, and was restored by incubation of these microsomes with egg yolk phospholipids. The extent of decrease in this phosphohydrolase activity in the detergent-exposed microsomes (with rupture of translocase activity) also varied depending on the detergent used (Triton X-114 or taurocholate). When 66% of the phosphatidylinositol on the membrane was hydrolyzed by phosphatidylinositol-specific phospholipase C of B. thuringiensis, the inhibition of glucose-6-P phosphohydrolase activity without detergent was very small. Although the inhibition of enzyme activity with detergent was apparently greater than that without detergent, the enzyme activity was stimulated by the breakdown of phosphatidylinositol when the enzyme activity was measured at lower concentration (0.5 mM) of substrate, glucose-6-P. The latency of mannose-6-P phosphohydrolase, a plausible index of microsomal integrity, remained above 70% after the hydrolysis of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol. The results show that the glucose-6-phosphatase system requires microsomal phospholipids for its integrity, suggesting that there exists a close relation between phosphatidylinositol and glucose-6-P translocase.     

The purification of a detergent-soluble glucose-6-phosphatase from rat liver.

A highly active and soluble glucose-6-phosphatase has been purified to near homogeneity from rat liver. Successful purification has been initiated by covalent labeling of the enzyme in native rat liver microsomes with pyridoxal 5'-phosphate and NaBH4, followed by solubilization of the microsomes with Triton X-100, chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sephacel and a second chromatography step on hydroxyapatite. The final enzyme preparation obtained was approximately 700-fold purified over the activity of starting microsomes. As judged by SDS/PAGE the purified glucose-6-phosphatase is composed of a single protein with a molecular mass of 35 kDa. The present work demonstrates that the purified glucose-6-phosphatase must be arranged in the native microsomal membrane so that it is accessible to pyridoxal 5'-phosphate from the cytoplasmic side.     

Alterations of the microsomal glucose-6-phosphatase system evoked by ferrous iron- and haloalkane free-radical-mediated lipid peroxidation

Alterations of catalytic activities of the microsomal glucose-6-phosphatase system were examined following either ferrous iron- or halothane (CF3CHBrCl) and carbon tetrachloride (CCl4) free-radical-mediated peroxidation of the microsomal membrane. Enzyme assays were performed in native and solubilized microsomes using either glucose 6-phosphate or mannose 6-phosphate as substrate. Lipid peroxidation was assessed by the amounts of malondialdehyde equivalents formed. Regardless of whether the experiments were performed in the presence of NADPH/Fe3+, NADPH/CF3CHBrCl, or NADPH/CCl4, with the onset of lipid peroxidation, mannose-6-phosphatase activity of the native microsomes increased immediately, while further alterations in catalytic activities were only detectable when lipid peroxidation had passed characteristic threshold values: above 2 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase activity of the native microsomes was lost, and at 10 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase and mannose-6-phosphatase activity of the solubilized microsomes started to decline. It is concluded that the latter alterations are due to an irreversible damage of the phosphohydrolase active site of the glucose-6-phosphatase system, while the changes observed at earlier stages of microsomal lipid peroxidation may also reflect alterations of the transporter components of the glucose-6-phosphatase system. Virtually no changes in the catalytic activities of the glucose-6-phosphatase system occurred under anaerobic conditions, indicating that CF3CHCl and CCl3 radicals are without direct damaging effect on the glucose-6-phosphatase system. Further, maximum effects of carbon tetrachloride and halothane on lipid peroxidation and enzyme activities were observed at an oxygen partial pressure (PO2) of 2 mmHg, providing additional evidence for the crucial role of low PO2 in the hepatotoxicity of both haloalkanes.     

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.     

Protease inhibitors but not proteases inhibit the glucose-6-phosphatase of native rat liver microsomes.

Controlled proteolytic digestion by trypsin or bacterial proteases limited to the cytosolic side of the native microsomal membrane is not efficient to inhibit glucose-6-phosphate hydrolysis. Modification of the microsomes with deoxycholate prior to protease treatment is prerequisite to allow accessibility of the integral protein and inhibition of enzyme activity. Glucose-6-phosphatase of native microsomes, however, is rapidly inactivated by micromolar concentrations of TPCK as well as TLCK. In deoxycholate-modified microsomes both reagents do not affect glucose-6-phosphate hydrolysis. These results indicate that in the native, intact microsomal membrane glucose-6-phosphatase is not accessible to proteolytic attack from the cytoplasmic surface. The putative inhibitory effect of some trypsin or bacterial protease preparations on glucose-6-phosphatase of native microsomes observed most possibly is a result of contaminating agents as TPCK or TLCK.     

The phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system is a 36.5-kilodalton polypeptide

The phosphohydrolase component of the microsomal glucose-6-phosphatase system has been identified as a 36.5-kDa polypeptide by 32P-labeling of the phosphoryl-enzyme intermediate formed during steady-state hydrolysis. A 36.5-kDa polypeptide was labeled when disrupted rat hepatic microsomes were incubated with three different 32P-labeled substrates for the enzyme (glucose-6-P, mannose-6-P, and PPi) and the reaction terminated with trichloroacetic acid. Labeling of the phosphoryl-enzyme intermediate with [32P]glucose-6-P was blocked by several well-characterized competitive inhibitors of glucose-6-phosphatase activity (e.g. Al(F)-4 and Pi) and by thermal inactivation, and labeling was not seen following incubations with 32Pi and [U-14C]glucose-6-P. In agreement with steady-state dictates, the amount of [32P]phosphoryl intermediate was directly and quantitatively proportional to the steady-state glucose-6-phosphatase activity measured under a variety of conditions in both intact and disrupted hepatic microsomes. The labeled 36.5-kDa polypeptide was specifically immunostained by antiserum raised in sheep against the partially purified rat hepatic enzyme, and the antiserum quantitatively immunoprecipitated glucose-6-phosphatase activity from cholate-solubilized rat hepatic microsomes. [32P]Glucose-6-P also labeled a similar-sized polypeptide in hepatic microsomes from sheep, rabbit, guinea pig, and mouse and rat renal microsomes. The glucose-6-phosphatase enzyme appears to be a minor protein of the hepatic endoplasmic reticulum, comprising about 0.1% of the total microsomal membrane proteins. The centrifugation of sodium dodecyl sulfate-solubilized membrane proteins was found to be a crucial step in the resolution of radiolabeled microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

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.     

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.     

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 multi- functional enzyme glucose-6-phosphatase (d-glucose-6-phosphatase 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 K_I 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.     

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.     

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.     

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)     

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.     

In vitro modification of cholesterol content of rat liver microsomes. Effects upon membrane 'fluidity' and activities of glucose-6-phosphatase and fatty acid desaturation systems

The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver.     

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.