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)     

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.     

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.     

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

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

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.     

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

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

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

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

The 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.     

Rapid kinetics of liver microsomal glucose-6-phosphatase. Evidence for tight-coupling between glucose-6-phosphate transport and phosphohydrolase activity.

Rapid kinetics of both glucose-6-P uptake and hydrolysis in fasted rat liver microsomes were investigated with a recently developed fast-sampling, rapid-filtration apparatus. Experiments were confronted with both the substrate transport and conformational models currently proposed for the glucose-6-phosphatase system. Accumulation in microsomes of 14C products from [U-14C]glucose-6-P followed biexponential kinetics. From the inside to outside product concentrations, it could be inferred that mostly glucose should accumulate inside the vesicles. While biexponential kinetics are compatible with the mathematical predictions of a simplified substrate transport model, the latter fails in explaining the "burst" in total glucose production over a similar time scale to that used for the uptake measurements. Since the initial rate of the burst phase in untreated microsomes exactly matched the steady-state rate of glucose production in detergent-treated vesicles, it can be definitely concluded that the substrate transport model does not describe adequately our results. While the conformational model accounts for both the burst of glucose production and the kinetics of glucose accumulation into the vesicles, it cannot explain the burst in 32Pi production from [32P]glucose-6-P measured under the same conditions. Since the amplitude of the observed bursts is not compatible with a presteady state in enzyme activity, we propose that a hysteretic transition best explains our results in both untreated and permeabilized microsomes, thus providing a new rationale to understand the molecular mechanism of the glucose-6-phosphatase system.     

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.     

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

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

N-Bromoacetylethanolamine phosphate as a probe for the identification of a liver microsomal glucose-6-phosphate transporter peptide in rats and Ehrlich ascites tumor-bearing mice

Hepatic microsomal glucose-6-phosphatase is a multicomponent system composed of substrate/product translocases and a catalytic subunit. Previously we (Foster et al. (1996) Biochim. Biophys. Acta 12, 244-254) demonstrated that N-bromoacetylethanolamine phosphate (BAEP) is a time-dependent, irreversible inhibitor of glucose-6-phosphate hydrolysis in intact but not disrupted microsomes. We proposed that BAEP manifests its inhibitory effect by binding with a glucose-6-phosphate translocase protein of the glucose-6-phosphatase system. Here we provide additional evidence that BAEP inhibits glucose-6-phosphate transport in microsomal vesicles and utilize [(32)P]BAEP as an affinity label in the identification of a glucose-6-phosphate transport protein. In this study, we identify 51-kDa rat and mouse liver microsomal proteins involved in glucose-6-phosphate transport into and out of microsomal vesicles by utilizing (1) an Ehrlich ascites tumor-bearing mouse model, which displays a decreased sensitivity to the time-dependent inhibitory effect of BAEP, and (2) another glucose-6-phosphate translocase inhibitor, tosyl-lysine chloromethyl ketone, in conjunction with [(32)P]BAEP as an affinity label.     

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.     

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.     

Use of protein-mediated lipid exchange in the study of membrane-bound enzymes. The lipid dependence of glucose-6-phosphatase.

The ability of liver lipid-exchange proteins to introduce foreign phospholipids into microsomes was used in a study of the lipid dependence of glucose-6-phosphatase. Supplementation of intact rat liver and hepatoma microsomes with exogeneous aminophospholipids prevents the decline of glucose-6-phosphatase activity during incubation, whereas the introduction of exogeneous phosphatidylcholine has no protective effect. On the contrary with deoxycholate-disrupted hepatoma microsomes, introduction of additional phosphatidylcholine causes activation while phosphatidylethanolamine has only little effect. The results are explained by assuming that the transport unit and the catalytic moiety of the glucose-6-phosphatase system have different lipid requirements, the activity of the former protein depending mainly on phosphatidylethanolamine and phosphatidylserine and that of the catalytic protein depending on phosphatidylcholine. In deoxycholate-disrupted liver microsomes (in which both the glucose-6-phosphatase activity and the phosphatidylcholine content are much higher than in hepatoma microsomes) incubation with phosphatidylcholine and lipid-exchange proteins alters neither the phospholipid composition nor the enzyme activity. THis suggests that the diminished activity of glucose-6-phosphatase in hepatomas may be partly due to a low level of phosphatidylcholine.     

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

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

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.     

Kinetic and immunologic evidence for the absence of glucose-6-phosphatase in early human chorionic villi and term placenta.

The existence of the enzyme glucose-6-phosphatase (G6Pase) in early and term human placenta was investigated by comparing the characteristics of placental microsomal glucose 6-phosphate (G6P) hydrolytic activity and liver G6Pase. Placental microsomes exhibited similar apparent Km values for G6P and beta-glycerophosphate in intact and deoxycholate-treated microsomes, heat stability at acidic pH, low latency of mannose 6-phosphate hydrolysis, very low activity of pyrophosphate: glucose phosphotransferase, and undetectable [U-14C]G6P transport into the placental microsomes, all of which indicated that specific G6Pase activity does not exist in placenta. Immunological evidence of the absence of both 36.5 kDa and T2 proteins, which represent the G6Pase catalytic protein and the phosphate/pyrophosphate transporter protein, respectively, confirmed that early and term human placenta are devoid of the multicomponent G6Pase enzyme.