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)

A method is described for the incorporation of a microsomal rat liver fraction into polyacrylamide films without significant loss of its glucose-6-phosphatase activity. The enzymatic activity was completely lost when the films were prepared with ammonium persulfate as initiator of the polymerization as previously described for alkaline phosphatase, but modification of this method showed that about 90% of the glucose-6-phosphatase activity could be retained. The enzyme in the films prepared with the new method was completely inhibited by alloxan, HgCl2, and preincubation in 0.05 M acetate buffer (pH 5.0) at 37 degrees C, as determined biochemically. Similar results were obtained for the enzyme in films determined histochemically according to the lead method of Wachstein and Meisel. In this respect the behavior of the incorporated enzyme is similar to that in suspension. Films fixed with 1.5% glutaraldehyde showed rapid inactivation of glucose-6-phosphatase. There was good correlation between the biochemical and histochemical activity determined after fixation. A method to embed polyacrylamide films in Epon for electron-microscopical investigation is also described. Dimethyl sulfoxide was used as the dehydrating agent instead of ethanol/acetone.
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PMID:Cytochemical model system for microsomal rat liver glucose-6-phosphate. 18 Jan 74

The histochemical detection of glucose-6-phosphatase (G-6-Pase) in neurons of the CNS has been confirmed at the level of electron microscope. Both glucose-6-phosphate (G-6-P) and alpha-glycerophosphate (alpha-gP) can be used as substrates to localize the reaction product of this enzyme, which we have found in all cell types of the cerebral cortex, cerebellum and brain stem. The reaction was most prominent in large neurons, such as the Purkinje cells of the cerebellum and the pyramidal cells of the cerebral cortex. This is due to their extensive content of rough and smooth endoplasmic reticulum, the ultrastructural sites of G-6-Pase activity. It was possible to measure quantitatively the hydrolysis of G-6-P and alpha-gP in brain homogenates and also in microsomal fractions, the biochemical correlate of the cytochemically demonstrable activity. These results call for a reappraisal of the previous biochemical evidence, which negates the existence of brain G-6-Pase, and consequently a reassessment of current concepts pertaining to the metabolic regulation of brain glucose.
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PMID:Cytochemical localization of glucose-6-phosphatase activity in the central nervous system of the rat. 18 19

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.
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PMID:Microsomal membrane permeability and the hepatic glucose-6-phosphatase system. Interactions of the system with D-mannose 6-phosphate and D-mannose. 18 83

Iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate (p-MB) and HgCl2 were tested as inhibitors of microsomal glucose-6-phosphatase. Iodoacetamide had no effect at 2 mM. N-ethylmaleimide inhibited only crude, but not purified microsomal preparations (M2) or crude microsomes exposed to deoxycholate. 14C-labelled N-ethylmaleimide was not bound by the M2 protein fraction. p-MB inhibited all types of preparations and the inhibition was not counteracted by detergent. A more detailed study was carried out with the purified M2 fraction (specific activity: 2-4 mumoles Pi/min/mg protein). Glucose-6-phosphate hydrolysis was inhibited 50% by 5 X 10(-5) M p-MB. The inhibition was completely reversible by dithiothreitol except when the enzyme was pre-incubated with p-MB in the absence of substrate. Then p-MB accelerated the temperature-dependent inactivation of glucose-6-phosphatase. Binding studies showed that around 3 mumoles 14C-p-MB were incorporated into 100 mg M2 protein regardless of the concentration of mercurial in the incubation mixture. That is, over a 25 fold range of p-MB concentration, causing up to 80% inhibition of enzyme activity, no difference was seen in the amount of labelled p-MB which was irreversibly bound to M2 protein. Kinetically p-MB behaved like a reversible inhibitor and this was confirmed by dilution experiments. Several compounds, including some amino acids, antagonized the inhibition by p-MB. The order of effectiveness was EDTA greater than barbital greater than tryptophan greater than histidine greater than lysine greater than other amino acids. Glycine, Tris and urea were ineffective competitors of p-MB inhibition. Double reciprocal plots showed that the Km for glucose-6-phosphate was increased and the Vmax reduced in the presence of p-MB. HgCl2 was a more effective inhibitor than p-MB with a Ki of 6 X 10(-6) M. We conclude that a reaction of p-MB with M2 sulfhydryls does not play a part in the inhibition of enzyme activity. It is suggested that p-MB may interact with one or more amino acid side chains in such a way that enzyme conformation is altered.
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PMID:The effect of p-hydroxymercuribenzoate and congeners on microsomal glucose-6-phosphatase. 18 75

A primary objective of the present study has been to determine the changes which occur in Rana catesbeiana liver organelle membranes during thyroxine-induced metamorphosis. To this end, enzyme and cytochrome profiles were determined for mitochondria, microsomes, and nuclear membrane fractions isolated from livers of R. catesbeiana tadpoles which had been fasted for 6 days at 15 +/- 0.5 degrees and then immersed in thyroxine, 2.6 X 10(-8) M, for periods of up to 12 days at 23.5 +/- 0.4 degrees. The ratio of total succinate-cytochrome c reductase activity in the initial homogenate fraction to the total activity of this mitochondrial "marker" enzyme recovered in the final mitochondrial fraction remained constant, approximately 0.5, throughout the course of thyroxine treatment; however, after a 3- to 4-day latency the mitochondrial protein mass recovered per unit mass of initial homogenate protein was found to increase significantly (approximately 2-fold by Day 10 of thyroxine treatment). A similar increase was also observed in the yield of microsomal, but not nuclear membrane, protein mass as a function of thyroxine treatment. Prolonged thyroxine treatment (12 days) resulted in approximately 50% decreases in tadpole liver homogenate and microsomal NADH-cytochrome c reductase specific activities; in contrast, mitochondrial and nuclear membrane NADH-cytochrome c reductase specific activities were not altered under the same conditions. In addition, homogenate and microsomal NADPH-cytochrome c reductase specific activities were found to have increased significantly after 12 days of thyroxine treatment; however, the specific activity of NADPH-cytochrome c reductase in the mitochondrial fraction was unchanged. It was also observed that thyroxine treatment resulted in increases in homogenate and microsomal glucose-6-phosphatase specific activities, whereas the mitochondrial as well as nuclear membrane glucose-6-phosphatase specific activities remained unchanged. Furthermore, in contrast to homogenate and mitochondrial monoamine oxidase specific activities, which decreased 30 and 40%, respectively, as a consequence of thyroxine treatment (12 days), the succinate-cytochrome c reductase and oligomycin-sensitive Mg2+ ATPase specific activities determined for these fractions increased significantly. In all instances, changes as a result of thyroxine treatment in membrane-localized homogenate or organelle enzyme specific activities were apparent only after a 3- to 4-day initial latent period. The in vitro effects of thyroxine (10(-10) - 10(-5) M) on the membrane-localized enzyme activities examined in this study were either negligible or, as in the case of mitochondrial succinate-cytochrome c reductase and microsomal NADH-cytochrome c reductase, opposite to the changes observed in response to in vivo thyroxine treatment, with the exception of microsomal NADPH-cytochrome c reductase activity which was enhanced approximately 2-fold by 10(-5) M thyroxine...
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PMID:Alterations in enzyme and cytochrome profiles of Rana catesbeiana liver organelles during thyroxine-induced metamorphosis. Changes in membrane-localized phosphohydrolases, oxidoreductases, and cytochrome levels in response to in vivo thyroxine administration. 18 3

Since infections with Schistosoma mansoni cause marked histopathological changes in the liver of the host, the effect of this infection on the hepatic drug-metabolizing function was investigated. Severity of Schistosomiasis was determined by worm counts, duration of infection, egg counts and liver weight increases. To overcome difficulties in homogenizing the livers of infected animals, preincubation of the squashed tissues with collagenase and hyaluronidase was used to prepare homogenates. Key component enzyme activities of the hepatic microsomal drug-metabolizing enzyme system (NADPH-cytochrome c reductase and cytochrome P-450) as well as the representative drug-metabolism activities (aminopyrine N-demethylase, aniline hydroxylase, and benzpyrene hydroxylase) were measured for the whole liver and found to be markedly reduced. However, the measurement of microsomal marker enzyme activities (cytochrome b5 and glucose-6-phosphatase) showed significant elevation. To obtain more precise information about the effect of the schistosome infection on the hepatic drug-metabolizing enzyme system, the total activities of microsomal drug-metabolizing enzymes were related to the total microsomal marker enzyme activities in the homogenate.
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PMID:Effect of Schistosoma mansoni infection on the hepatic drug-metabolizing capacity of mice. 18 61

The presented paper describes the role of enzyme histochemistry in cell biological investigations. In the first chapter a general discussion has been given about enzyme histochemistry as a connecting link between biochemistry and morphology. The methods available for determination of enzymes in a particular cell or cell compartment have been reviewed. In this respect the characteristics of enzyme histochemistry have been discussed. Furthermore, attention has been paid to the possibilities and limitations of enzyme histochemistry. In chapter two a comparison has been made between histochemically judged and biochemically determined enzyme activities. Some fundamental differences between the biochemical and the histochemical approach in cell biological investigations are dealt with. To correlate histochemically and biochemically determined enzyme activities, a description has been given of the application of histochemical methods on isolated fractions and sucrose-ficoll gradients of these fractions. Several experimental results are described concerning the question whether a relation exists between histochemically and biochemically determined activities of respectively alkaline phosphatase, glucose-6-phosphatase, 5'-nucleotidase and 3ss-hydroxysteroid dehydrogenase. From these results the conclusion could be drawn that in general a good correlation exists between histochemically judged activity per volume (area X thickness) and biochemically determined activity per gram tissue. In chapter three the role of enzymes as markers of cellular particles and as parameters of metabolic pathways is described. Histochemical methods are available for most marker enzymes. Only activities of key enzymes can be regarded as parameters of metabolic pathways. The distribution in sucrose-ficoll gradients of enzymes, regarded as markers of mitochondria, lysosomes, endoplasmic reticulum and plasma membranes has been given. The changes occur ing under different experimental conditions for a number of marker enzymes in rat liver are described. Attention has been given to the contibution of enzyme histochemistry in the study of the heterogeneity of mitochondria, the dual localization of some (lysosomal) enzymes, the complexity of the microsomal fraction, the function of the Golgi apparatus and the heterogeneity and function of plasma membranes. Based on these results and on literature findings the possible role of some marker enzymes in cell metabolism has been discussed. In chapter four problems coherent with species and sex differences in enzyme activities are described. The interpretation of histochemical and biochemical results in view of these differences is discussed. Enzymes characteristic for a given cell type -3ss-hydroxysteroid dehydrogenase in steroid producing cells, ATP-ase in liver plasma membrane surrounding the bile canaliculi - do show less variations between species and sexes than enzymes not directly involved in specialized functions...
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PMID:Enzyme histochemistry as a link between biochemistry and morphology. 18 46

Noradrenaline-storing granules, a mitochondrial fraction and a microsomal fraction of bovine splenic nerve trunks were prepared by differential centrifugation. These particulate fractions were characterized by their noradrenaline content, succinate dehydrogenase and glucose-6-phosphatase activity. In the presence of ATP-Mg2+ all three fractions accumulated 45Ca2+ during incubation with 0.1 mM 45 CaCl2, buffered with potassium phosphate or glycylglycine (pH 7.5; 28 degrees C). The accumulated 45 Ca2+ was not removable by EGTA, and the uptake was absent at 0 degrees C or after destruction of the particles by sonication. The behaviour of the 45 Ca2+ -uptake into all three fractions against varying ATP-concentrations, metabolic inhibitors (pentachlorophenol, desaspidine, 2,4-dinitrophenol, N-ethylmaleimide, p-chloromercuribenzoate, sodium azide, amobarbital) and drugs (phenoxybenzamine, verapamil, prenylamine, reserpine, bretylium, phentolamine) was studied. Under nearly all conditions there were differences between the 45 Ca2+ -uptake into mitochondria and that into microsomes, which suggests two distinct uptake processes. The 45 Ca2+ -uptake into the granule fraction behaved intermediate between the two other fractions under many conditions, but not under all. Therefore, it is not possible to explain the 45 Ca2+ -uptake into the granule fraction as being due to contamination with mitochondria and microsomes; an inherent ATP-Mg2+ -dependent 45Ca2+ -uptake into the nerve granules must be postulated, which is not directly coupled with the noradrenaline transport into these particles. A particulate fraction (14000-100000 g), containing noradrenaline granules, was prepared from the vas deferens of the rat. Incubation with 5 X 10(-6) M (-)-noradrenaline and 0.1 mM 45Ca2+ showed that the particles of this fraction take up noradrenaline and 45Ca2+. The uptake of both was dependent on ATP-Mg2+. The ATP-Mg2+ -dependent uptake of both noradrenaline and 45Ca2+ was substantially reduced in the corresponding tissue fraction prepared from denervated vasa deferentia.
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PMID:Ca2+ -uptake into noradrenaline-storing granules of bovine splenic nerves. 18 27

The transverse distribution of enzyme proteins and phospholipids within microsomal membranes was studied by analyzing membrane composition after treatment with proteases and phospholipases. Upon trypsin treatment of closed microsomal vesicles, NADH- and NADPH-cytochrome c reductases as well as cytochrome b5 were solubilized or inactivated, while cytochrome P-450 was partially inactivated. When microsomes were exposed to a concentration of deoxycholate which makes them permeable to macromolecules but does not disrupt the membrane, the detergent alone was sufficient to release four enzymes: nucleoside diphosphatase, esterase, beta-glucuronidase, and a portion of the DT-diaphorase. Introduction of trypsin into the vesicle lumen inactivated glucose-6-phosphatase completely and cytochrome P-450 partially. The rest of this cytochrome, ATPase, AMPase, UDP-glucuronyltransferase, and the remaining 50% of DT-diaphorase activity were not affected by proteolysis from either side of the membrane. Phospholipase A treatment of intact microsomes in the presence of albumin hydrolyzed all of the phosphatidylethanolamine, phosphatidylserine, and 55% of the phosphatidylcholine. From this observation, it was concluded that these lipids are localized in the outer half of the bilayer of the microsomal membrane; Phosphatidylinositol, 45% of the phosphatidylcholine, and sphingomyelin are tentatively assigned to the inner half of this bilayer. It appears that the various enzyme proteins and phospholipids of the microsomal membrane display an asymmetric distribution in the transverse plane.
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PMID:Enzyme and phospholipid asymmetry in liver microsomal membranes. 19 Feb 41

A procedure for the determination of liver microsomal glucose-6-phosphatase is described. Homogenization and ultracentrifrigation were used to prepare a precipitate whose character was defined by monitoring the desire enzyme activity which serves as a marker. Activity of the enzyme was determined by means of a sensitive colorimetric reaction for the product, inorganic phosphate. Non-enzymatic hydrolysis problems with the substrate are minimized in this procedure by the masking action of citrate. The final heteropoly blue color appears to be considerably sensitized by interaction of phosphomolybdous ion with arsenite. The stability of the relatively labile enzyme was ensured by chelating any metals present with ethylene diamine tetraacetic acid. The overall results obtained by the procedure appear to be useful as an aid in the diagnosis of Type I glycogenosis, a glycogen storage disease called Von Gierke's disease.
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PMID:Determination of liver microsomal glucose-6-phosphatase. 19 25


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