Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.9 (glucose-6-phosphatase)
 3,081 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatic microsomal glucose-6-phosphatase (Glc-6-P'ase) is a complex multicomponent system containing at least three transport proteins, in addition to the catalytic subunit and a Ca2+ binding regulatory protein. The transport proteins have been designated T1 the glucose-6-phosphate transport protein, T2 a phosphate/pyrophosphate transport protein and T3 a glucose transport protein. Diagnosis of the genetic deficiencies of these transport proteins at present requires a complex kinetic analysis of the Glc-6-P'ase system as a whole. Here we describe the progress to date in our attempts to identify, purify and clone each transport protein with the ultimate aim of isolating specific cDNA probes for each transport protein which can be used for the diagnosis of types 1b, 1c and the putative 1d glycogen storage diseases.
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PMID:Identification, purification and genetic deficiencies of the glucose-6-phosphatase system transport proteins. 839 41

Previous studies have demonstrated that the hepatotoxin carbon tetrachloride rapidly promotes lipid peroxidation and inhibits microsomal calcium sequestration, microsomal glucose-6-phosphatase activity and cytochrome P-450. Due to its profound effects on lipid peroxidation, we have examined the oral administration of 2.5 ml/kg carbon tetrachloride on the urinary excretion of the lipid metabolites formaldehyde, malondialdehyde, acetaldehyde and acetone. Urine samples were collected up to 48 h after treatment. The urinary metabolites were identified and quantitated by gas chromatography-mass spectrometry and high-pressure liquid chromatography. Time-dependent increases in the urinary excretion of the four metabolites were observed after carbon tetrachloride administration. At 48 h after treatment, the increases in the excretion of malondialdehyde, formaldehyde, acetaldehyde and acetone were approximately 55, 78, 57 and 268%, respectively, relative to control values. The data were expressed in nanomoles per kilogram body weight per 4.5 h. The results clearly demonstrate that carbon tetrachloride increases the urinary excretion of four lipid metabolites which may serve as noninvasive biomarkers of xenobiotic-induced lipid peroxidation.
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PMID:Carbon-tetrachloride-induced urinary excretion of formaldehyde, malondialdehyde, acetaldehyde and acetone in rats. 841 71

The purpose of the present study was to develop a technique to identify, isolate and partially purify these membrane bound compartments for further characterizations of their Ca2+ transport and storage mechanisms. We 45Ca(2+)-loaded the agonist-sensitive Ca2+ stores in rat pancreatic acini. The loading was accomplished by first depleting the stores with carbachol stimulation followed by the addition of 45Ca2+ and atropine to the extracellular media. After homogenization of the 45Ca(2+)-loaded acini, subcellular fractions were resolved on sucrose and Nycodenz gradients. 45Ca2+ fluxes were minimized during these procedures by inclusion in the media of LaCl3. Five subcellular fractions were identified that specifically accumulated 45Ca2+ after carbachol stimulation. Electron microscopic observations of the fractions demonstrated that three of the fractions consisted of rough membrane vesicles; that one consisted of a mixture of rough and smooth membrane vesicles; and that one consisted of smooth membrane vesicles. All fractions were enriched in glucose-6-phosphatase. All 5 fractions demonstrated ATP dependent 45Ca2+ uptake. By Western blot analysis, all fractions contained calnexin, p58, sarcoplasmic reticulum type Ca(2+)-ATPase, and IP3 receptor. These results demonstrated that the 45Ca(2+)-loading technique can be used to isolate and characterize distinct compartments of the agonist-sensitive Ca2+ store in the pancreatic acinar cell.
Cell Calcium 1995 Nov
PMID:Isolation of subcellular agonist-sensitive calcium stores from the pancreatic acinar cell. 858 65

The dependence of microsomal glucose-6-phosphatase (G-6-Pase) activity on Ca2+ as well as the membrane lipid microviscosity was studied by the effect of Ca(2+)-channel blockers (namely verapamil and nifedipine), Ca(2+)-ionophore, A23187 and pyrene excimer formation. Channel blockers depressed the G-6-Pase and Ca(2+)-ATPase while the ionophore increased these activities. Dimethyl sulfoxide, a known membrane surface active agent showed no change. Ca(2+)-uptake into the membrane has expectedly been lowered by the channel blockers while the ionophores facilitated the ion flux. Excimer formation of the fluorescent probe, pyrene as an indicator of increased membrane fluidity, and microviscosity calculated from there on, showed that Ca(2+)- and lipid microenvironment in the membrane significantly influenced the activity of G-6-Pase. Membrane lipid composition such as phospholipid/cholesterol molar ratio which also indicates an increased membrane fluidity is markedly increased with the ionophore but decreased with the channel blockers, while protein/phospholipid ratio remained unchanged. Microsomal G-6-Pase is a multicomponent multifunctional protein. It is argued that Ca2+ may play the role of an obligatory cofactor not only for the hydrolysis of G-6-P (catalytic part of the enzyme) but also involved in the regulation of substrate and product transport in or out of the endoplasmic reticulum lumen.
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PMID:Effect of Ca(2+)-channel blockers, Ca(2+)-ionophore and increased pyrene excimer formation on the microsomal glucose-6-phosphatase. 871 49

Synaptic plasma membrane (SPM) vesicles represent a membrane fraction very useful in studying non-vesicular neurotransmitter release. The procedure described here to isolate SPM vesicles from a crude synaptosomal fraction of sheep brain cortex is quick, simple (ultracentrifugation in a discontinuous density gradient of dextran T110), and combines a high yield (130 micrograms/g brain) with a satisfactory grade of purification. The preparation of SPM vesicles consists of vesicles (approximately 0.54 +/- 0.8 micron diameter) delimited by a single membrane with the native orientation. We are able to ascertain these characteristics on the basis of morphology studies (electron microscopy observations), enzyme activities (Na+/K(+)-ATPase, Ca2+/Mg(2+)-ATPase, acetylcholinesterase and glucose-6-phosphatase), biochemical composition (lipid and protein analysis) and the tetrodotoxin sensitivity of the veratridine-induced gamma-aminobutyric acid (GABA) release. Isolating the SPM vesicles by the proposed procedure permits manipulating the ionic gradients across the membrane by changing the ion concentrations on either side or by utilizing specific ionophores. The vesicles retain their various activities, including their capacity for neurotransmitter uptake and release assays for at least 3 months, when preserved at -70 degrees C. Furthermore, the vesicles permit depicting the electrochemical gradients across the membranes into chemical and electrical components. We describe the use of the tetraphenylphosphonium cation (TPP+) to dissipate the membrane potential (delta psi) of the vesicles, while preserving ionic gradients. The characteristics of the lipid-soluble cation TPP+ allows a massive inflow of this cation into vesicular compartments and a consequent depolarization.
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PMID:Membrane potential manipulation in synaptic plasma membrane vesicles for studying neurotransmitter uptake and release. 938 41

S 5627 is a synthetic analogue of chlorogenic acid. S 5627 is a potent linear competitive inhibitor of glucose 6-phosphate (Glc-6-P) hydrolysis by intact microsomes (Ki = 41 nM) but is without effect on the enzyme in detergent- or NH4OH-disrupted microsomes. 3H-S 5627 was synthesized and used as a ligand in binding studies directed at characterizing T1, the Glc-6-P transporter. Binding was evaluated using Ca2+-aggregated microsomes, which can be sedimented at low g forces. Aside from a modest reduction in K values for both substrate and S 5627, Ca2+ aggregation had no effect on glucose-6-phosphatase (Glc-6-Pase). Scatchard plots of binding data are readily fit to a simple "two-site" model, with Kd = 21 nM for the high affinity site and Kd = 2 microM for the low affinity site. Binding to the high affinity site was competitively blocked by Glc-6-P (Ki = 9 microM), whereas binding was unaffected by mannose-6-phosphate, Pi, and PPi and only modestly depressed by 2-deoxy-D-glucose 6-phosphate, a poor substrate for Glc-6-Pase in intact microsomes. Thus the high affinity 3H-S 5627 binding site fits the criteria for T1. Permeabilization of the membrane with 0.3% (3-[(chloramidopropyl)-dimethylammonio]-1-propanesulfonate) activated Glc-6-Pase and broadened its substrate specificity, but it did not significantly alter the binding of 3H-S 5627 to the high affinity sites or the ability of Glc-6-P to block binding. These data demonstrate unequivocally that two independent Glc-6-P binding sites are involved in the hydrolysis of Glc-6-P by intact microsomes. The present findings are the strongest and most direct evidence to date against the notion that the substrate specificity and the intrinsic activity of Glc-6-Pase in native membranes are determined by specific conformational constraints imposed on the enzyme protein. These data constitute compelling evidence for the role of T1 in Glc-6-Pase activity.
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PMID:Direct evidence for the involvement of two glucose 6-phosphate-binding sites in the glucose-6-phosphatase activity of intact liver microsomes. Characterization of T1, the microsomal glucose 6-phosphate transport protein by a direct binding assay. 949 46

The levels of iron, zinc, and calcium in liver as well as serum, together with the enzymatic activities of gamma-glutamyl transferase (GGT, EC 2.3.2.2) and glucose-6-phosphatase (G-6-Pase, EC 3.1.3.9) in liver, were critically monitored over various periods in male Swiss albino mice bearing Dalton's lymphoma (DL), a transplantable ascites-producing tumor. Both hepatic and serum contents of iron, zinc, and calcium were found to be maximally elevated (p < 0.001) on day 15 after tumor transplantation as compared with their contents in normal animals. There was a gradual increase in the activity of GGT in liver in lymphoma-bearing mice in comparison with their normal counterparts, which showed a maximum peak (p < 0.001) on day 15, followed by a continuous and sharp fall. Hepatic G-6-Pase activity was found to decrease continuously throughout the progression of lymphoma as compared with its levels to normal animals. Tumor-cell counts in peritoneal lymph fluids of mice containing DL yielded a maximum count of 155.7 x 10(3) cells/mm3 on day 15. A significant correlation was observed among the levels of different metals, enzymatic activities, and tumor-cell counts at different periods of study. From these results, it can be concluded that the metals studied may have a role in initiating and controlling cellular proliferations, through their effects on modulating the activities of the possibly preneoplastic and neoplastic marker enzymes named above.
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PMID:Alterations in total iron, zinc, and calcium levels and their influence on the hepatic activities of gamma-glutamyl transferase and glucose-6-phosphatase in the host bearing transplantable murine lymphoma. 958 38

Microsomes prepared from three rat tissues were examined for their ability to import glucose-6-phosphate (G-6-P). Microsomes from liver, which possess a high level of glucose-6-phosphatase activity, were compared with those from cerebral cortex and cardiac muscle, which are not involved in the export of glucose and in which glucose-6-phosphatase activity is relatively low. In all three, a selective permeability to G-6-P was detected by light scattering. However, the sugar-phosphate specificity of the transport process differed. G-6-P was able to enhance ATP-dependent Ca2+ sequestration in all three types of microsomes. In addition, enzymatic detection of G-6-P after the rapid filtration of microsomes determined that, in the absence of Ca2+ and ATP, a level of intramicrosomal G-6-P approaching a passive equilibrium with the extramicrosomal G-6-P concentration was rapidly achieved in all three tissues. However, under conditions in which Ca2+ was being actively accumulated, the intramicrosomal levels of G-6-P exceeded the equilibrium value by three- to fourfold. This enhanced sequestration was not observed in the presence of Ca2+ or ATP alone or in the presence of a Ca2+ ionophore or an inhibitor of the microsomal Ca2+ ATPase. These data are consistent with a selective import pathway into the endoplasmic/sarcoplasmic reticulum for G-6-P independent of glucose-6-phosphatase activity. In addition, they suggest an alternate explanation for the enhanced sequestration of Ca2+ by the endoplasmic/sarcoplasmic reticulum of intact cells seen when extracellular glucose is increased.
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PMID:Glucose-6-phosphate and Ca2+ sequestration are mutually enhanced in microsomes from liver, brain, and heart. 960 62

We sought a rapid and non-ultracentrifugal method of recovering large amounts of highly pure rough endoplasmic reticulum (RER) membranes from livers. By substantially modifying a 20-year-old calcium precipitation technique, we obtained a RER fraction from rat liver and established its high degree of purity by quantitating classic membrane markers for different subcellular organelles. This RER fraction is highly enriched in four known proteins (or enzyme activities) required for lipoprotein assembly: apolipoprotein B, microsomal triglyceride transfer protein, acyl CoA:diacylglycerol acyltransferase, and acyl CoA:cholesterol acyltransferase, when compared to two classical RER markers, RNA and glucose-6-phosphatase. From one 10-12 g rat liver, we recover ten to twelve RER pellets of 1.5-1.6 cm in diameter containing approximately 110-125 mg of total protein, about half of which is sodium carbonate-releasable. By electron microscopy, these large RER pellets from rat livers are homogeneously comprised largely of non-vesiculated short strips of ribosome-rich membranes. This novel technique for isolating RER membranes from liver may provide a useful tool for future studies on the assembly of apolipoprotein B-containing lipoproteins as well as for research focused on mechanisms of secretory and membrane protein translation, translocation, and folding.
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PMID:A rapid calcium precipitation method of recovering large amounts of highly pure hepatocyte rough endoplasmic reticulum. 1035 46

The efficacy of Tiron and calcium disodium EDTA in the treatment of experimental beryllium intoxication was investigated in rats. Beryllium nitrate was administered intramuscularly (50 mg kg(-1)) once only, provoking duration-dependent changes. Maximum changes were recorded after a 7-day regimen. Considerable inhibition was recorded in protein and glycogen contents, as well as in the activity of alkaline phosphatase, glucose-6-phosphatase, acid phosphatase and lipid peroxidation. These parameters were restored considerably with chelation therapy, but comparatively Tiron offered better protection. These findings were further confirmed by atomic adsorption spectrophotometry. Tiron was found to be significantly more effective than CaNa(2)EDTA in reducing the beryllium concentration in the liver, kidney and lungs.
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PMID:Beryllium-induced toxicity and its prevention by treatment with chelating agents. 1094 6


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