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 Morris 7777 hepatoma on microsomal glucose-6-phosphatase latent activity

The change in the activity of several hepatic enzymes during hepatocarcinogenesis suggests a pattern of dedifferentiation. This category of enzymes includes glucose-6-phosphatase and gamma-glutamyltranspeptidase (GGT). A detailed kinetic analysis of microsomal glucose-6-phosphatase activity revealed that both the translocase and phosphohydrolase activities were markedly reduced in Morris 7777 hepatoma transplanted in male Buffalo rats. In addition, the activity of the translocase component increased 2.4-fold, while the phosphohydrolase activity decreased 1.6-fold in the liver of tumor-bearing animals. GGT activity in the host liver was not effected by the presence of the tumor. These results suggest differences in the effect of Morris 7777 hepatoma on: the phosphohydrolase and translocase activities of microsomal glucose-6-phosphatase and the sensitivity of glucose-6-phosphatase and GGT activities in the host liver.     

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.     

Role of polyfunctional glucose-6-phosphatase in the liver carbohydrate metabolism of the developing chick embryo

Glucose-6-phosphatase was shown to be polyfunctional in the liver of the developing chick embryo. Changes in the activity of glucose-6-phosphate phosphohydrolase did not correlate with the rate of gluconeogenesis. The activity of this enzyme increased from the 16th to the 20th day of embryogenesis. The activities of pyrophosphate-glucose phosphotransferase, carbamyl-phosphate-glucose phosphotransferase did not change during embryogenesis. The ratio of the activities of phosphohydrolase and phosphotransferases was characterized by the predominance of the phosphohydrolase activity. The values of latency of phosphohydrolase and phosphotransferases did not correlate with the rate of gluconeogenesis. Glucose-6-phosphate phosphohydrolase was found not only in the microsomal, but in the nuclear fraction as well. KM(G6P) of the enzyme of the nuclear fraction differed from KM of the microsomal enzyme.     

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.     

Hysteretic behavior of the hepatic microsomal glucose-6-phosphatase system.

Carbamyl-P:glucose and PPi:glucose phosphotransferase, but not inorganic pyrophosphatase, activities of the hepatic microsomal glucose-6-phosphatase system demonstrate a time-dependent lag in product production with 1 mM phosphate substrate. Glucose-6-P phosphohydrolase shows a similar behavior with [glucose-6-P] less than or equal to 0.10 mM, but inorganic pyrophosphatase activity does not even at the 0.05 or 0.02 mM level. The hysteretic behavior is abolished when the structural integrity of the microsomes is destroyed by detergent treatment. Calculations indicate that an intramicrosomal glucose-6-P concentration of between 20 and 40 microM must be achieved, whether in response to exogenously added glucose-6-P or via intramicrosomal synthesis by carbamyl-P:glucose or PPi:glucose phosphotransferase activity, before the maximally active form of the enzyme system is achieved. It is suggested that translocase T1, the transport component of the glucose-6-phosphatase system specific for glucose-6-P, is the target for activation by these critical intramicrosomal concentrations of glucose-6-P.     

Multiple thermal discontinuities in glucose-6-phosphatase activity.

The temperature dependence of glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase EC 3.1.3.9) was studied in rat liver and kidney microsomal fractions. Arrhenius plots were non-linear and showed four distinct discontinuities in enzyme activity over the temperature range 2-41 degrees C. The discontinuities occurred at approx. 39, 30, 20 and 12 degrees C in the liver and were similar to this in the kidney. Changes in the energy of activation for the enzyme were noted at approx. 20 degrees C in both tissues. The multiple discontinuities in glucose-6-phosphatase activity are viewed as a reflection of complex reorganization and/or change in physical state of the membrane components, primarily lipid.     

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].     

Effects of basic proteins of low molecular weight on the phosphohydrolase and phosphotransferase activities of microsomal glucose-6-phosphatase in adult monkey hepatocytes (author's transl)

The effects of histone 2A and some polycations on microsomal carbamylphosphate:D-glucose phosphotransferase and glucose-6-phosphate phosphohydrolase activities (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9), have been investigated. 1. Histone 2A and polycations activate the two enzymic activities. At a constant cation concentration, this activation increases with the number of cationic groups per molecule. 2. Activation by histone 2A is related to its fixation on microsomal membranes. This fixation varies with quantities of histones and pH. 3. The nature of the interactions between histones and microsomal membranes is shown to be electrostatic, probably between the cationic groups of histones and the anionic group of membranous lipids. 4. Kinetic analysis reveal that histone 2A increases the maximal reaction velocity but does not affect the apparent Michaelis constant values for the substrates. 5. The role played by the cationic groups of histone 2A on the microsomal glucose 6-phosphatase, is discussed.     

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.     

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.     

Different developmental changes in latency for two functions of a single membrane bound enzyme: glucose-6-phosphatase activities as a function of age.

(1). The capacity for the synthesis of glucose 6-phosphate from PPi and glucose as well as for glucose-6-P hydrolysis, catalyzed by rat liver microsomal glucose-6-phosphatase, increases rapidly from low prenatal levels to a maximum between the second and fifth day, then slowly decreases to reach adult levels. When measured in enzyme preparations optimally activated by hydroxyl ions, the maximum neonatal activities were 4--5-fold higher than in adult animals and several-fold higher than had previously been observed for the unactivated enzyme. (2) The latencies of two catalytic activities associated with the same membrane-bound enzyme show strikingly different age-related changes. The latency of PPi-glucose phosphotransferase activity reaches high levels (60--80% latent) soon after birth and remains high throughout life, while the latency of glucose-6-P phosphohydrolase decreases with age. The phosphohydrolase is 2--3 times more latent in the liver of the neonatal animal than in the adult. (3). The well established neonatal overshoot of liver glucose-6-phosphatase is almost entirely due to changes in the enzyme in the rough microsomal membranes. The enzyme activity in the rough membrane reaches a maximum and then decreases after day 2, while that in the smooth membrane is still slowly increasing. Despite the great differences in absolute specific activities and in the pattern of early enzyme development between the rough and smooth microsomes, enzyme latency in the two subfractions remains parallel, glucose-6-P phosphohydrolase being only slightly more latent, while PPi-glucose phospho-transferase is much more latent in smooth than in rough membranes throughout life. (4). Kidney glucose-6-P phosphohydrolase and PPi-glucose phosphotransferase activities were found to change in a parallel fashion with age, showing a small neonatal peak between days 2 and 7 before rising to adult levels. Kidney phosphotransferase activity, like that of liver, remained highly latent throughout life. In contrast to liver, the glucose-6-P phosphohydrolase of kidney did not show a characteristic decrease in latency with age and in the adult remained appreciably more latent than in liver. (5). An improved method was devised for the separation of smooth microsomes from liver homogenates.     

Subcellular localization of glucose-6-phosphatase in animal tissues.

1. Glucose-6-phosphatase (EC 3.1.3.9 D-glucose-6-phosphate phosphohydrolase) was found to be localized mainly in the endoplasmic reticulum (microsomal fraction) of all species of vertebrate liver tissue examined. 2. Hepatopancreas tissue from gastropod molluscs was found to be unique in showing the localization of glucose-6-phosphatase in the cytosol (soluble fraction).     

Nuclear membranes from mammalian liver. VI. Glucose-6-phosphatase in rat liver, a cytochemical and biochemical study

Activities of glucose-6-phosphatase (G-6-Pase) and other phosphatases were determined in nuclei, nuclear membrane and microsomal fractions and subfractions, and condensed chromatin isolated from the liver of adult, newly born and prenatal rats. The purity of the fractions was controlled by electron microscopic morphometry and by measurement of various marker enzymes. The specific G-6-Pase activity of the nuclear membranes was found to be about 60% that of the microsomes. However, when calculated on the basis of the phospholipid content, all fractions had similar activities. Determinations of G-6-Pase enrichments and recoveries were also made. The correspondence of the hydrolysing activities of glucose-6-phosphate, mannose-6-phosphate, and inorganic pyrophosphate, together with various phosphotransferases, showed the same association of the G-6-Pase with these enzymes in the nuclear envelope as in the microsomal membranes. G-6-Pase was also demonstrated in the fractions by cytochemistry, and the activity was localized alongside the cisternal surfaces of both, inner and outer, nuclear membrane. ‘Free’ inner nuclear membrane fragments contained also G-6-Pase. No activity was observed at the nuclear pore complexes. Both, nuclear and microsomal membranes revealed a parallel rapid perinatal increase of G-6-Pase activity climaxing at 23 to 28 h after birth. Triton-X-100 treatment of isolated nuclei, which was found not to selectively release outer nuclear membranes, resulted in a great decrease of G-6-Pase activity as well as in losses of membrane phospholipids. The results clarify the divergence of earlier reports concerning the presence of G-6-Pase in the perinuclear cisterna and add biochemical evidence to the morphologically derived view of the nuclear envelope as being a special form of the ER system.     

A pH-metrical method of determining glucose-6-phosphatase activity

A simple, rapid, and reproducible method of determining glucose-6-phosphatase activity is described. The glucose 6-phosphate hydrolysis is accompanied by the disappearance of the protons from the medium owing to a phosphate species pK change from 6.1 (in glucose 6-phosphate) to 6.9 (in inorganic phosphate). Alkalization is registered by a pH meter with a recorder. The method described in this paper may be used in routine determinations of glucose-6-phosphatase activity.     

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

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

Stimulation of glucose-6-phosphatase by polyamines is a membrane-mediated event

Discontinuities in Arrhenius plots occuring at 18° for both carbamyl-P:glucose phosphotransferase and glucose-6-P phosphohydrolase activities of D-glucose-6-P phosphohydrolase (EC 3.1.3.9) are either eliminated (phosphotransferase) or shifted (phosphohydrolase) by 1 mM spermidine, spermide, or putrescine. Since the observed discontinuity is due to physical changes in the microsomal membrane at 18°, alteration or elimination of the discontinuity upon the addition of polyamine is interpreted to be a result of charge neutralization occurring from the interaction of the polycation with the negative charges on the membrane surface.     

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.     

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.     

Role of microviscosity of the lipid phase of microsomal membranes in regulation of enzymatic activity of glucose-6-phosphatase under microsomal modifications in vivo and in vitro

The changes in microviscosity of the lipid phase of microsomal membranes under microsomal modification in vivo and in vitro were studied. It was shown that in intact microsome lipids there occur five thermo-induced structural transitions within the temperature range of 5--50 degrees. Delipidation of microsomes results in a shift in structural transitions temperature. Based on the literary and own data it was assumed that the breaks on the Arrhenius plots for glucose-6-phosphatase (EC 3.1.3.9) activity are due to phase-structural changes of microsomal lipids.