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.     

Evidence for changes in the conformational status of rat liver microsomal glucose-6-phosphate:phosphohydrolase during detergent-dependent membrane modification. Effect of p-mercuribenzoate and organomercurial agarose gel on glucose-6-phosphatase of native and detergent-modified microsomes

Comparative studies investigating influences of temperature and time of preincubation on the interactions of an organomercurial agarose gel and p-mercuribenzoate with glucose-6-phosphatase of native and Triton X-114-modified rat liver microsomes were carried out. The effect of p-mercuribenzoate on glucose 6-phosphate hydrolysis is a result of two processes, a moderate membrane perturbation connected with release of some latency and temperature- and time-dependent inhibition of the catalytic activity. Short-term preincubation with both organic mercurials at 37 degrees C is a necessary condition for the entire inhibition of the enzyme activity of native as well as of Triton X-114-modified microsomes. A binding site of the phosphohydrolase itself is accessible to p-mercuribenzoate and the phenyl mercury residue of the affinity gel from the cytoplasmic surface even in native microsomes. Kinetic analyses reveal a formally competitive mechanism of inhibition using native microsomes, but the kinetic picture changes to a noncompetitive pattern of Lineweaver-Burk plots when the inhibitor-loaded microsomes are modified optimally by Triton X-114. This behavior can be evaluated as the first convincing evidence for drastic changes of the conformational status of the phosphohydrolase during the membrane modification process. A combined conformational flexibility-substrate transport model characterizing the microsomal glucose-6-phosphatase as an integral channel-protein embedded within the hydrophobic interior of the membrane is proposed.     

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].     

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.     

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.     

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 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.     

Partial purification and characterization of lathosterol 5-desaturase from rat liver microsomes

The terminal oxidase of the NADH-dependent lathosterol 5-desaturation system was solubilized from rat liver microsomes with 2% Triton X-100, and partially purified approximately 18-fold with 19% yield after DEAE-cellulose and 6-aminohexyl-Sepharose column chromatography. The final enzyme preparation was free from other electron transfer components and phospholipids in microsomes, and the desaturation reaction was reconstituted with the following components: NADH, molecular oxygen, phospholipids and three proteins, i.e., NADH-cytochrome b5 reductase, cytochrome b5 and the terminal oxidase. Omission of one of these components led to an almost complete loss of the desaturase activity. Under the reconstitution conditions, the desaturase activity was significantly inhibited by potassium cyanide but was not affected by -SH reagents such as N-ethylmaleimide and dithiothreitol.     

Immunochemical evidence for the enzymatic difference of delta 6-desaturase from delta 9- and delta 5-desaturase in rat liver microsomes

The enzymatic properties of the three types of microsomal acyl-CoA desaturases, delta 6-, delta 9- and delta 5-desaturases, were immunologically compared using a monospecific antibody raised against the purified linoleoyl-CoA desaturase (delta 6-desaturase). By the double immunodiffusion technique, the anti-delta 6-desaturase antibody showed a single precipitin line to the purified delta 6-desaturase and microsomes treated with Triton X-100, but no line was observed with the partially purified delta 9-desaturase. The antibody even inhibited definitely delta 6-desaturase activity in microsomes, but neither stearoyl-CoA (delta 9-) nor eicosatrienoic acid (delta 5-) desaturations were inhibited. By these immunological investigations it was confirmed that terminal delta 6-desaturase is different enzyme from desaturases delta 9- and delta 5.     

Partial purification and characterization of collagen galactosyltransferase from chick embryos.

Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.     

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.     

Partial purification and characterization of beta-mannosyltransferase from suspension-cultured soybean cells

The beta-mannosyltransferase that catalyzes the synthesis of Man-beta-GlcNAc-GlcNAc-PP-dolichol from GDP-mannose and dolichyl-PP-GlcNAc-GlcNAc was solubilized from microsomes of suspension-cultured soybean cells by treatment with 1.5% Triton X-100 and was purified about 700-fold by chromatography on DEAE-cellulose, hydroxylapatite, and a GDP affinity column. The purified enzyme was reasonably stable in the presence of 20% glycerol and 0.5 mM dithiothreitol. The enzyme required either detergent (Triton X-100 or NP-40) or phospholipid for maximum activity, but the effects of these two were not additive. Thus, either phosphatidylcholine or Triton X-100 could give maximum stimulation. In terms of phospholipid stimulation, both the head group and the acyl chain appeared to be important since phosphatidylcholines with 18-carbon unsaturated fatty acids were most effective. The purified enzyme had a sharp pH optimum of 6.9-7.0 and required a divalent cation. Mg2+ was the best metal ion with optimum activity occurring at 6 mM, but Mn2+ was reasonably effective while Ca2+ was slightly stimulatory. The Km for GDP-mannose was calculated to be 1.7 X 10(-6) M and that for dolichyl-PP-GlcNAc-GlcNAc about 9 X 10(-6) M. The enzyme was inhibited by a number of guanosine nucleotides such as GDP-glucose, GDP, GMP, and GTP, but various uridine and adenosine nucleotides were without effect. The purified enzyme was apparently free of alpha-1,3-mannosyltransferase (and perhaps other mannosyltransferases) and dolichyl-P-mannose synthase since the only product seen from dolichyl-PP-GlcNAc-GlcNAc and GDP-mannose was Man-beta-GlcNAc-GlcNAc-PP-dolichol.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterization and partial purification of squalene-2,3-oxide cyclase from Saccharomyces cerevisiae.

The membrane nature of squalene oxide cyclase from Saccharomyces cerevisiae was investigated by comparing properties of the enzyme recovered from both microsomes and the soluble fraction of the yeast homogenate. The "apparent soluble" form and microsomal form of the enzyme were both stimulated by the presence of mammalian soluble cytoplasm and corresponded to one another in response to detergents Triton X-100 and Triton X-114. The observed strong dependence of the enzyme activity on the presence of detergents and the behavior of the enzyme after Triton X-114 phase separation were peculiar to a lipophilic membrane-bound enzyme. A study of the conditions required to extract the enzyme from microsomes confirmed the lipophilic character of the enzyme. Microsomes, exposed to ipotonic conditions to remove peripheral membrane proteins, retained most of the enzyme activity within the integral protein fraction. Quantitative dissociation of the enzyme from membranes occurred only if microsomes were treated with detergents (Triton X-100 or octylglucoside) at concentrations which alter membrane integrity. The squalene oxide cyclase was purified 140 times from yeast microsomes by (a) removal of peripheral proteins, (b) extraction of the enzyme from the integral protein fraction with octylglucoside, and (c) separation of the solubilized proteins by DEAE Bio-Gel A chromatography. Removal of the peripheral proteins seemed to be a key step necessary for obtaining high yields.     

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.     

Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC

The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C(12)E(8) and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca(2+)-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C(12)E(8) and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of the membrane-bound enzyme.     

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.     

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.     

Relationship between microsomal membrane permeability and the inhibition of hepatic glucose-6-phosphatase by pyridoxal phosphate.

Arion et al; (Arion, W. J., Wallin, B. K., Lange A. J., and Ballas, L. M. (1975) Mol. Cell. Biochem. 6, 75-83) propsed a model for glucose-6-phosphatase in which the substrate was transported across the microsomal membrane by a carrier before hydrolysis on the cisternal side. Evidence to support this model has been obtained by studying the inhibition of the enzyme by pyridoxal-P. Pyridoxal-P was a linear noncompetitive inhibitor of glucose-6-phosphatase (EC 3.1.3.9) in freshly isolated ("intact") microsomes from rat liver. Pyridoxol-P was a much less effective inhibitor and no inhibition was observed with pyridoxamine-P. When microsomes were subjected to nitrogen cavitation, treatment with solium deoxycholate, or glutaraldehyde fixation, the Km of glucose-6-phosphatase for glucose-6 P decreased from approximately 6 mM to approximately 2.5 mM; the corresponding change in the Vmax ranged from-10% to +40%. The same procedures decreased the inhibition of glucose-6-phosphatase by pyridoxal-P several-fold. No inhibition by pyridoxal-P was observed in a preparation of glucose-6-phosphatase purified approximately 20 fold (on the basis of Vmax) from micoromes. A nondialyzable inhibitor was apparently formed when intact microsomes were reacted with pyridoxal-P and NaBH4; this inhibition was also reversed by procedures which changed the kinetic properties of glucose-6-phosphatase.     

Inhibition by aldehydes as a possible further mechanism for glucose-6-phosphatase inactivation during CCl4-poisoning

Diffusable aldehydes are known to be produced during lipoperoxidative deterioration of unsaturated fatty acids. Malealdehyde (MLA) and 4-hydroxy-2,3-trans-penten-1-al (4-HPE) inhibit rat liver glucose-6-phosphatase activity in vitro. With MLA inhibition is significant at 0.25 mM concentration. With 4-HPE inhibition takes place at 0.5 mM. 1 mM MLA inhibited by about 89%, 6 mM 4-HPE by about 67%. Maximal inhibition is present as early as 5 min after addition of both aldehydes. Preincubation of aldehydes with 2 mM cysteine or glycine in the absence of microsomes almost completely prevents the inhibitory influence. Previous incubation of microsomes with 2 mM glutathione or 2 mM dithiothreitol or 2 mM cysteine affords a good protection towards the inhibitory action of the aldehydes; on the contrary, no protection is seen when microsomes are preincubated in the presence of either 2 mM glycine or asparagine. The total content of microsomes -SH groups is strongly decreased after incubation with 2mM malealdehyde.These results support the idea that the two aldehydes inhibit glucose-6-phosphatase mostly through interaction with protein -SH groups. The possibility that aldehydes derivated from the peroxidative decomposition of lipids may play a cooperative role in the inhibition of glucose-6-phosphatase occurring early after CCl₄-poisoning is discussed.