DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : II. Glucose-6-Phosphatase in Rough Microsomes

Electron microscope cytochemical localization of glucose-6-phosphatase in the developing hepatocytes of fetal and newborn rats indicates that the enzyme appears simultaneously in all the rough endoplasmic reticulum of a cell, although asynchronously within the hepatocyte population as a whole. To confirm that the pattern of cytochemical deposits reflects the actual distribution of enzyme sites, a method to subfractionate rough endoplasmic reticulum was developed. The procedure is based on the retention of the cytochemical reaction product (precipitated lead phosphate) within freshly prepared rough microsomes reacted in vitro with glucose-6-phosphate and lead ions. Lead phosphate increases the density of the microsomes which have glucose-6-phosphatase activity and thereby makes possible their separation from microsomes lacking the enzyme; separation is obtained by isopycnic centrifugation on a two-step density gradient. The procedure was applied to rough microsomes isolated from rats at several stages during hepatocyte differentiation and the results obtained agree with those given by cytochemical studies in situ. Before birth, when only some of the cells react positively for glucose-6-phosphatase, only a commensurate proportion of the rough microsome fraction can be rendered dense by the enzyme reaction. At the time of birth and in the adult, when all cells react positively, practically all microsomes acquire deposit and become dense after reaction. Thus, the results of the microsome subfractionation confirm the cytochemical findings; the enzyme is evenly distributed throughout all the endoplasmic reticulum of a cell and there is no regional differentiation within the rough endoplasmic reticulum with respect to glucose-6-phosphatase. These findings suggest that new components are inserted molecule-by-molecule into a pre-existing structural framework. The membranes are thus mosaics of old and new molecules and do not contain large regions of entirely "new" membrane in which all of the components are newly synthesized or newly assembled.     

Association of isolated bovine kidney cortex peroxisomes with endoplasmic reticulum

Close lateral membrane associations of peroxisomes with endoplasmic reticulum are a common feature in bovine kidney cortex epithelial cells. Isolated highly purified peroxisome preparations from this tissue showed a remarkable and persistent copurification of peroxisomal marker enzymes with small amounts (5%) of the microsomal reference enzymes esterase and glucose-6-phosphatase. Contamination with mitochondrial and lysosomal markers was negligible. Ultrastructural examination of such preparations revealed a peculiar association of vesicles or short tubular segments with the peroxisomal membrane. Short electron dense crossbridges seemed to maintain their structural association. The cytochemical localization of glucose-6-phosphatase in peroxisome-associated membrane structures confirmed their derivation from endoplasmic reticulum. The metabolic significance of such structural peroxisome-endoplasmic reticulum associations is discussed.     

Studies on vinblastine-induced autophagocytosis in mouse liver. IV. Origin of membranes

The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Electron microscopical demonstration of glucose-6-phosphatase in native cryostat sections fixed with glutaraldehyde through semipermeable membranes.

The cytochemical demonstration of glucose-6-phosphatase (G6Pase) activity in native cryostat sections fixed with glutaraldehyde through semipermeable membranes is superior to conventional methods with regard to exact localization and lack of inactivation and diffusion of the enzyme, together with simultaneous excellent preservation of the tissue fine structure. In rat liver not only hepatocytes but also many bile duct epithelia and endothelia of arterioles and venules show a marked G6Pase activity in the membranes of the endoplasmic reticulum including the nuclear envelope.     

Ultrastructural localization of enzymatic activity during spermiogenesis in two phytophagous bugs (Hemiptera: Pentatomidae)

Ultrastructural cytochemical techniques were used for the localization of phosphatases and oxidases in spermatid and spermatozoon of the phytophagous bugs Acrosternum aseadum and Nezara viridula (Hemiptera: Pentatomidae). Acid phosphatase was found mainly in the trans most portion of the Golgi complex, and in the acrosome of spermatozoon. Glucose-6-phosphatase was located in the endoplasmic reticulum, trans portion of the Golgi complex and in the acrosome of spermatids. The axoneme showed activity of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase. This observation supports the idea that various phosphates may play some role in spermatid differentiation. Indeed, the presence of cytochrome oxidase activity was only shown in the mitochondrial cristae of early spermatids, suggesting also the participation of this enzyme during spermatid differentiation of this insect.     

Electron microscopical demonstration of glucose-6-phosphatase in native cryostat sections fixed with glutaraldehyde through semipermeable membranes

Summary The cytochemical demonstration of glucose-6-phosphatase (G6Pase) activity in native cryostat sections fixed with glutaraldehyde through semipermeable membranes is superior to conventional methods with regard to exact localization and lack of inactivation and diffusion of the enzyme, together with simultaneous excellent preservation of the tissue fine structure. In rat liver not only hepatocytes but also many bile duct epithelia and endothelia of arterioles and venules show a marked G6Pase activity in the membranes of the endoplasmic reticulum including the nuclear envelope. This work was kindly supported by the Deutsche Forschungsgemeinschaft An erratum to this article is available at .     

Loss of endoplasmic reticulum membrane integrity: an image analysis of the glucose-6-phosphatase system in human hepatocyte.

Histochemical and cytochemical methods induce a loss of endoplasmic reticulum (ER) membrane integrity in hepatocytes. In order to evaluate the degree of ER membrane integrity, glucose-6-phosphatase (G6P-A) was localized in light and electron microscopy using glucose-6-phosphate (G6P) and mannose-6-phosphate (M6P) as substrates. In case of ER membrane alteration, M6P diffuses inside the ER and is hydrolysed by a non-specific phosphohydrolase. G6P and M6P hydrolysis was quantified with image analysis methods. In light microscopy, the ratio of reaction of M6P hydrolysis/G6P hydrolysis gave 75% of non specific reaction. In electron microscopic study this ratio was about 30%. These results showed that enzyme localization methods in electron microscopy produced less ER membrane alteration than light microscopic methods.     

Cytochemical localization of enzymes in the spermatid and the spermatozoon of Culex quinquefasciatus say (Diptera : Culicidae)

Ultrastructural cytochemical techniques were used for the localization of phosphatases in spermatid and spermatozoon of the mosquito, Culex quinquefasciatus (Diptera : Culicidae). Acid phosphatase was found mainly in the trans-most portion of the Golgi complex. Thiamine pyrophosphotase was preferentially located in the cis-most portion of the Golgi complex. Glucose-6-phosphatase was located in the endoplasmic reticulum and cisternae of the transition zone between the endoplasmic reticulum and the Golgi complex. The complex membrane of the anterior acrosomal region and the axoneme showed acid phosphatase activity. Reaction products indicating the presence of acid phosphatase, thiamine pyrophosphatase, and glucose-6-phosphatase, were observed on the spermatozoon surface at the head and tail regions. These observations support the idea that various phosphatases may play some role in spermatid differentiation.     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum an nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Summary Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum and nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.     

Glucose-6-phosphatase distribution in isolated and cultured adult rat hepatocytes

The cytochemical localization of glucose-6-phosphatase (G6Pase) and its biochemical quantification were studied in isolated and cultured adult rat parenchymal cells. Appropriate technical conditions were chosen to assume adequate ultrastructural preservation and retention of enzyme activity. Isolated hepatocytes separated by collagenase perfusion were shortly fixed in glutaraldehyde and entrapped in a pellet of fibrin. Frozen sections, 50 microns in thickness were incubated for cytochemical demonstration of G6Pase, in a slightly modified Wachstein-Meisel medium. Hepatocytes in culture, fixed for 1 min in glutaraldehyde, were impregnated in a 10% cryoprotective glycerol solution and quickly frozen in liquid nitrogen at -170 degrees C in order to induce penetration of the substrate. In these conditions, a homogeneous distribution of the enzyme was observed in both isolated and cultured cells. The cytochemical reaction appears continuous in the smooth and rough endoplasmic cisternae and in the nuclear envelope. Lead phosphate deposits, although evenly distributed, are reduced in intensity after 48 h culture. Biochemical determinations reveal the presence of a high specific enzymatic activity in isolated cells (108 nmolP/min/mg proteins), which decreases in culture, respectively to 70 and 50% of the original value, after 24 and 48 h culture. G6Pase induction by glucagon was obtained after 48 and 72 h in culture.     

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : I. Glucose-6-Phosphatase Distribution In Situ

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid endoplasmic reticulum differentiation (4 days before birth-1 day after birth) was studied by electron microscope cytochemistry. Techniques were devised to insure adequate morphological preservation, retain glucose-6-phosphatase activity, and control some other possible artifacts. At all stages examined the lead phosphate deposited by the cytochemical reaction is localized to the endoplasmic reticulum and the nuclear envelope. At 4 days before birth, when the enzyme specific activity is only a few per cent of the adult level, the lead deposit is present in only a few hepatocytes. In these cells a light deposit is seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, when the specific activity of glucose-6-phosphatase is approximately equal to that of the adult, nearly all cells show a positive reaction for the enzyme and, again, the deposit is evenly distributed throughout the entire endoplasmic reticulum. By 24 hr postparturition all of the rough endoplasmic reticulum, and in addition the newly formed smooth endoplasmic reticulum, contains heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6-phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although asynchronously in the hepatocyte population as a whole. In addition, the enzyme appears throughout the entire smooth endoplasmic reticulum as the membranes form during the first 24 hr after birth. The results suggest a lack of differentiation within the endoplasmic reticulum with respect to the distribution of glucose-6-phosphatase at the present level of resolution.     

Histochemical enzyme pattern of microfilaria of Setaria cervi

The present study deals with the histochemical localization of glucose-6-phosphatase, malic dehydrogenase and aldolase in the microfilaria of Setaria cervi. Marked activity of glucose-6-phosphatase was observed in the cephalic cells, excretory and anal pores, G-cells and Innenk?rper. Malic dehydrogenase activity was noted throughout the body (including cuticle) of the microfilaria except for Innenk?rper. Intense aldolase activity was observed in the excretory pore and G-cells only. Muscle cells and anal pore were negative for this enzyme.     

Frog hepatocyte modifications induced by seasonal variations: a morphological and cytochemical study.

A correlated morphological and cytochemical approach was employed to study frog hepatocytes in different periods of their annual cycle, including the natural hibernating period. There were considerable changes in the distribution and organization of hepatic glycogen in different phases of the annual cycle, and distribution of organelles as well. The most striking findings were glycogen storage during the prehibernation and hibernation phases, followed by drastic glycogen depletion. Cytochemical staining of a number of enzymes (succinate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, paranitrophenyl phosphatase, acid phosphatase, and glucose-6-phosphatase) involved in a variety of metabolic pathways, showed various cytoplasmic localizations and differences in intensity of the reaction products as a function of seasonality. Morphological and cytochemical data were interpreted as evidencing different functional requirements during seasonal changes in the frog.     

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.     

Effects of hormones on the activity of glucose-6-phosphatase in primary cultures of rat hepatocytes

Although the activity of glucose-6-phosphatase in rat liver is altered markedly following the administration of a variety of hormones in vivo, it is not certain whether the hormones act directly on the hepatocyte. To study this problem hepatocytes were isolated by a collagenase-perfusion technique and cultured on collagen gel/nylon mesh membranes. The activity of glucose 6-phosphatase in cells cultured with fetal calf serum and with Dulbecco's modified Eagle's medium or Leibovitz L-15 medium decreased to less than 10-30% of the activity in freshly isolated cells by 96 h. However, when L-15 plus newborn or fetal calf serum was supplemented with glucagon (10(-6)M), epinephrine (10(-6)M), triiodothyronine (10(-6)M), and dexamethasone (10(-5)M) (L-15-GETD), the activity of glucose-6-phosphatase was maintained so that, after 144 h, the activity was at least 80% of that detected in freshly isolated cells. In cells cultured in L-15 plus serum for 72 or 96 h and then in L-15-GETD, glucose-6-phosphatase increased 30-50% over that in control cultures after 24 h. Insulin, which decreases glucose-6-phosphatase activity when administered to intact animals, also decreased the glucose-6-phosphatase activity in cultured hepatocytes to 20-50% of that in controls.     

A protein in crude cytosol regulates glucose-6-phosphatase activity in crude microsomes to regulate group size in Dictyostelium

Dictyostelium discoideum form groups of approximately 2 x 10(4) cells. The group size is regulated in part by a negative feedback pathway mediated by a secreted multipolypeptide complex called counting factor (CF). The CF signal transduction pathway involves CF-repressing internal glucose levels by increasing the K(m) of glucose-6-phosphatase. Little is known about how this enzyme is regulated. Glucose-6-phosphatase is associated with microsomes in both Dictyostelium and mammals. We find that the activity of glucose-6-phosphatase in crude microsomes from cells with high, normal, or low CF activity had a negative correlation with the amount of CF present in these cell lines. In crude cytosols (supernatants from ultracentrifugation of cell lysates), the glucose-6-phosphatase activity had a positive correlation with CF accumulation. The crude cytosols were further fractionated into a fraction containing molecules greater than 10 kDa (S>10K) and molecules less than 10 KDa (S<10K). S>10K from wild-type cells strongly repressed the activity of glucose-6-phosphatase in wild-type microsomes, whereas S>10K from countin(-) cells (cells with low CF activity) significantly increased the activity of glucose-6-phosphatase in wild-type microsomes by decreasing K(m). The regulatory activities in the wild-type and countin(-) S>10Ks are heat-labile and protease-sensitive, suggesting that they are proteins. S<10K from both wild-type and countin(-) cells did not significantly change glucose-6-phosphatase activity. Together, the data suggest that, as a part of a pathway modulating multicellular group size, CF regulates one or more proteins greater than 10 KDa in crude cytosol that affect microsome-associated glucose-6-phosphatase activity.     

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.     

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].