Ultrastructural localization of glucose-6-phosphatase in renal glomerulus of the adult dog

The ultrastructural distribution of glucose-6-phosphatase activity was investigated in renal glomeruli of adult dogs by electron-microscopic cytochemistry. The enzymatic activity was found mainly in the parietal epithelial cells of Bowman's capsule. Weaker activity occurred in visceral epithelial cells. No activity was found in either the endothelial or the mesangial cells. Strong activity of glucose-6-phosphatase was commonly found in the nuclear envelope, and occasionally in the rough endoplasmic reticulum. The distribution of the enzyme and its functional significance are discussed in relation to previously reported data.     

Ultrastructural localization of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium of rat

The effect of ovarian hormones on the activities of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium was studied in immature and ovariectomized rats, using ultracytochemical techniques. Comparative studies were done on normal rats at the luteal phase and on day 14 of pregnancy. Various vaginal cells show different degrees of response to progesterone and diethylstilbestrol (DES) with regard to glucose-6-phosphatase activity. Intense glucose-6-phosphatase activity was observed in the cisternae of granular endoplasmic reticulum (rER), Golgi saccules and vesicles, and nuclear envelope of both basal cells and stromal cells of progesterone treated rats, whereas in the basal cells and stromal cells of DES-treated and control animals the enzyme was totally lacking. Detectable glucose-6-phosphatase activity was also observed, however, in the rER cisternae and Golgi complex of keratohyalin-secreting squamous intermediate cells of the vaginal epithelium of DES-treated rats. Alkaline phosphatase was also found on the limiting membranes of secretory granules of mucocytes in animals at the luteal phase and during pregnancy. DES and progesterone in the doses used did not affect alkaline phosphatase activity in the rat vagina. Overall, progesterone enhances glucose-6-phosphatase activity in basal cells of the rat vagina prior to completion of mucification. Alkaline phosphatase was found in all cells involved in mucin secretion.     

Ultrastructural localization of intestinal glucose-6-phosphatase activity during the postnatal development of the mouse

Summary The development of the endoplasmic reticulum (ER) and the ultrastructural localization of glucose-6-phosphatase activity have been studied in the proximal jejunum and distal ileum during the postnatal period. One day after birth, the amount and the repartition of ER in the jejunal enterocytes are similar to that observed in postweaning period. In the following days an extensive proliferation of SER is noted in the supranuclear zone of the absorbing cells. From day 7 till postweaning period a gradual decrease of the amount of SER is observed and after weaning, the ultrastructure of the enterocytes is similar to that in the adult mouse enterocytes. At all time, a positive reaction for G-6-Pase activity is observed in the cisternae of the endoplasmic reticulum and in the nuclear envelope. In the distal ileum, the SER is poorly developed one day after birth. During the first two weeks, the ER increases but no extensive proliferation of SER can be noted as in the jejunum. The G-6-Pase activity can be visualized in the rough and smooth endoplasmic reticulum as well as in the nuclear envelope. It appears that the proliferation of SER could be interpreted as the morphologic expression of an increased G-6-Pase activity.This work was supported by research grant from the Medical Research Council of CanadaD. Ménard, Ph. D. is «Chercheur-boursier du Conseil de la recherche en santé du Québec»     

Ultrastructural localization of intestinal glucose-6-phosphatase activity during the postnatal development of the mouse

The development of the endoplasmic reticulum (ER) and the ultrastructural localization of glucose-6-phosphatase activity have been studied in the proximal jejunum and distal ileum during the postnatal period. One day after birth, the amount and the repartition of ER in the jejunal enterocytes are similar to that observed in postweaning period. In the following days an extensive proliferation of SER is noted in the supranuclear zone of the absorbing cells. From day 7 till postweaning period a gradual decrease of the amount of SER is observed and after weaning, the ultrastructure of the enterocytes is similar to that in the adult mouse enterocytes. At all time, a positive reaction for G-6-Pase activity is observed in the cisternae of the endoplasmic reticulum and in the nuclear envelope. In the distal ileum, the SER is poorly developed one day after birth. During the first two weeks, the ER increases but no extensive proliferation of SER can be noted as in the jejunum. The G-6-Pase activity can be visualized in the rough and smooth endoplasmic reticulum as well as in the nuclear envelope. It appears that the proliferation of SER could be interpreted as the morphologic expression of an increased G-6-Pase activity.     

Ultrastructural localization of glucose-6-phosphatase activity in proximal convoluted tubule cells of rat kidney

Summary The ultrastructural localization of glucose 6-phosphatase activity was investigated in the proximal convoluted tubule cells of the rat kidney. The reaction product for the enzyme activity was present in the endoplasmic reticulum and nuclear envelope, as reported for the hepatic enzyme and others, but was absent from the brush border, plasma membrane and other organelles. The metabolic significance of the association of this enzyme with the endoplasmic reticulum and the role of the enzyme in the active reabsorption and transport of glucose in the renal tubules are discussed.     

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).     

Latency of inosine-5'-diphosphatase in microsomes isolated from rat liver.

The latency of inosine-5'-diphosphatase has been studied in microsomes isolated from rat liver. The appearance of latent activity was the result of an increase in the Vmax of the enzyme. This was observed when assays were carried out in the presence of sodium deoxycholate, after microsomes were treated wtih phospholipase C, or at pH 10.3 and after microsomes were subjected to nitrogen cavitation. The apparent Km of inosine-5'-diphosphatase for IDP was unchanged when microsomes were treated with phospholipase C or at pH 10.3 after both these treatments approximately 85% of the enzyme remained bound to the membrane. In contrast, when microsomes were treated with phospholipase C or at pH 10.3 after both these treatments approximately 85% of the enzyme remained bound to the membrane. In contrast, when microsomes were treated with sodium deoxycholate or subjected to nitrogen cavitation, approximately 75% of the inosine-5'-diphosphatase activity was released from the membrane, and the apparent Km of the enzyme for IDP increased 4- and 2-fold, respectively. Microsomal cisternae were loaded with lead phosphate by incubation with glucose-6-P and Pb2+, and the release of this lead phosphate following the addition of EDTA to the medium was determined to estimate the permeability of the microsomal membrane. When microsomes were treated with sodium deoxycholate, phospholipase C, or at alkaline pH, the microsomal membrane became almost completely permeable to EDTA under conditions where there was little or no increase in the activity of inosine-5'-diphosphatase. Microsomes were treated at pH 10.3 and then adjusted slowly to pH 7.5. The activity of inosine-5'-diphosphatase decreased to the same activity observed in untreated preparations. The results seem of exclude the possibility that latent inosine-5'-diphosphatase activity is the result of an increased permeability of the membrane to IDP. They are, however, consistent with the presence of a noncompetitive inhibitor of the enzyme in the microsomal membrane.     

Cytochemical localization of glucose-6-phosphatase activity in cerebral endothelial cells

Glucose-6-phosphatase (G6Pase) activity, with glucose-6-phosphate and mannose-6-phosphate as substrates, was examined by cytochemistry in capillary and arteriole endothelial cells of the mouse brain. G6Pase activity was observed ultrastructurally in the lumen of the nuclear envelope and endoplasmic reticulum (ER) of these cells. The reactive ER and nuclear membrane appeared to be in continuity. Nucleoside diphosphatase activity, also a marker for the ER in some cell types, was not seen within the ER of the cerebral microvasculature. The ER of arterioles and capillaries did not bind lead nonspecifically when incubated in a substrate-free medium. Speculation is raised concerning the involvement of G6Pase in glucose metabolism of cerebral endothelial cells and in making blood-borne glucose available to brain parenchyma.     

Histochemical localization of glucose-6-phosphatase and 5-nucleotidase in the digestive system of Ophiocephalus Channa punctatus.

The histochemical localization of G6Pase and 5-Nase in the digestive system of Ophiocephalus (Channa) punctatus was studied. The highest activities of these enzymes were found in the liver. Appreciable activity was also found in the anterior intestine (duodenum) and pyloric caeca. The activity faded toward the middle and posterior intestine and rectum. In the stomach the activity was moderate. The activity of 5-Nase was weaker than that of G6Pase. In the stomach the enzymes were localized in the mucosa and gastric glands. The absorptive columnar epithelial cells were the major sites of localization in the intestine. The goblet cells were negative. The G6Pase activity was associated with the cytoplasm, while the 5-Nase activity was found in the cell membranes and the nuclei.     

Cytochemical localization of glucose-6-phosphatase in rabbit mononuclear phagocytes during differentiation

Cytochemical and biochemical investigations have revealed glucose-6-phosphatase (G-6-Pase) activity in Kupffer cells of the liver. To determine whether other mononuclear phagocytes are also reactive for G-6-Pase, rabbit bone marrow, blood, and alveolar macrophages were tested for G-6-Pase by a modified Wachstein-Meisel method and prepared for electron microscopy. Some mononuclear phagocytes from all three tissues were intensely reactive; others were unreactive. In promonocytes, monocytes, and alveolar macrophages, reaction product for the enzyme was localized throughout all cisternae of the endoplasmic reticulum (ER) and the perinuclear cisternae, but it was absent from the Golgi complex, lysosomes, and occasional smooth tubular channels. These results indicate that mononuclear phagocytes at all stages of development contain cytochemically demonstrable G-6-Pase and that the distribution of the enzyme is not altered during their differentiation from immature cells in the bone marrow to mature macrophages in the lung.     

Perinatal development of glucose-6-phosphatase and fructose-1,6-diphosphatase activities in pig liver

The activity of glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphosphatase (FDPase) was determined in the homogenate of the liver of 69 pig fetuses during the last third of gestation (80th to 114th day), 47 piglets from birth to 4 weeks old (suckling period) and to slaughter pigs. G6Pase is evident in fetal liver at an early date and raises steadily during gestation. In newborn piglets, the enzyme activity increases rapidly during the first hours of life and remains at this high level during the first week of life. Afterwards the enzyme activity returns to birth level, which exists also in pigs at slaughtering. The activity of FDPase is constant during the fetal period. After birth enzyme activity rises at a lower rate than the G6Pase during the first week of life. This level remains constant during the suckling period and increases thereafter until the time of slaughtering of pigs. The role of hormones in the perinatal development of these enzymes is described. Probably, thyroxine causes the prenatal increase of the activity of both the enzymes. The rapid postnatal rise of G6Pase activity may be induced by the high level of hydrocortisone at parturition, and furthermore, glucagon may have a permissive effect.     

Molecular pathology of glucose-6-phosphatase

It was known in the 1950s that hepatic microsomal glucose-6-phosphatase plays an important role in the regulation of blood glucose levels. All attempts since then to purify a single polypeptide with glucose-6-phosphatase activity have failed. Until recently, virtually nothing was known about the molecular basis of glucose-6-phosphatase or its regulation. Recent studies of the type 1 glycogen storage diseases, which are human genetic deficiencies that result in impaired glucose-6-phosphatase activity, have greatly increased our understanding of glucose-6-phosphatase. Glucose-6-phosphatase has been shown to comprise at least five different polypeptides, the catalytic subunit of glucose-6-phosphatase with its active site situated in the lumen of the endoplasmic reticulum; a regulatory Ca2+ binding protein; and three transport proteins, T1, T2, and T3, which respectively allow glucose-6-phosphate, phosphate, and glucose to cross the endoplasmic reticulum membrane. Purified glucose-6-phosphatase proteins, immunospecific antibodies, and improved assay techniques have led to the diagnosis of a variety of new type 1 glycogen storage diseases. Recent studies of the type 1 glycogen storage diseases have led to a much greater understanding of the role and regulation of each of the glucose-6-phosphatase proteins.     

Stimulation by polyamines of carbamylphosphate:glucose phosphotransferase and glucose-6-phosphate phosphohydrolase activities of multifunctional glucose-6-phosphatase.

The effects of added polyamines on carbamylphosphate (carbamyl-P):glucose phosphotransferase and glucose-6-phosphate (Glc-6-P) phosphohydrolase activities of rat hepatic D-Glc-6-P phosphohydrolase (EC 3.1.3.9) of intact and detergent-treated microsomes have been investigated. With the former preparation, in the presence of 1.4 mM phosphate substrate and 90 mM D-glucose (phosphotransferase), 1 mM spermine, spermidine, and putrescine activated Glc-6-P phosphohydrolase 67%, 57%, and 35%, respectively. Carbamyl-P:glucose phosphotransferase, under comparable conditions, was activated 57%, 34%, and 18%. NH+4 (0.25--5.0 mM) produced at best but a minor activation (0--14%), while poly(L-lysine) (Mr = 3400; degree of polymerization 16) equimolar relative to other polyamines with respect to ionized free amino groups activated the hydrolase 358% and the transferase 222%. Treatment of microsomes with the detergent deoxycholate reduced, but did not abolish, polyamine-induced activation. The stimulatory effects of polyamines persisted in the presence of excess catalase, indicating their independence from H2O2 formation; and were eliminated in the presence of Ca2+. Kinetic analysis revealed that all tested polyamines decreased the apparent Michaelis constant values for carbamyl-P and Glc-6-P, but had no effect on the Km for glucose. Poly(L-lysine) increased the V value for both Glc-6-P phosphohydrolase and apparent V values for phosphotransferase extrapolated to infinite concentrations of either carbamyl-P or glucose. The other tested polyamines elevated only this last velocity parameter. It is proposed that a major mechanism by which polyamines activate glucose-6-phosphatase-phosphotransferase is through their electrostatic interactions with phospholipids of the membrane of the endoplasmic reticulum of which this enzyme is a part. Conformational alterations thus induced may in turn affect catalytic behavior. It is suggested that polyamines, or similar positively charged peptides, might participate in the cellular regulation of synthetic and hydrolytic activities of glucose-6-phosphatase.     

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)