UNIPROT:Q8NEX9 - FACTA Search

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Query: UNIPROT:Q8NEX9 (reductase)
26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Homogenates of porcine corpus luteum were subjected to fractionation by differential-rate centrifugation or sucrose density gradient fractionation, with or without pretreatment with digitonin. Fractions of each gradient were assayed for a number of markers characteristic of the major intracellular organelles and cell-surface membranes, and for progesterone content. The majority of the progesterone content of homogenates of porcine corpus luteum was associated with a low-density particulate fraction which equilibrated at a buoyant density of 1.07-1.09 g/cm3. Pretreatment with digitonin increased the buoyant density of the progesterone-enriched fraction markedly (to 1.13-1.15 g/cm3) without causing release of steroid. The density distributions of progesterone content in control and digitonin-treated luteal gradient fractions were quite distinct from those of the major intracellular organelles and luteal cell-surface membranes. However, NADH-cytochrome C reductase activity (but not other endoplasmic reticulum markers) was also enriched in this fraction. The results suggest that most of the progesterone of the porcine corpus luteum is associated with a unique particulate fraction which is enriched in digitonin-reactive lipids and NADH-cytochrome C reductase activity.
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PMID:Subcellular fractionation of the porcine corpus luteum: sequestration of progesterone in a unique particulate fraction. 339 91

The transport of phosphate into jejunal endoplasmic reticulum vesicles isolated from suckling and adolescent rats was investigated using a rapid filtration technique. Intestinal endoplasmic reticulum from both ages were enriched with NADPH cytochrome-C-reductase whereas other markers for brush border, basolateral, mitochondrial, and Golgi apparatus were impoverished. Phosphate uptake represented an energy-dependent process as evident by more than 80% decrease in uptake values at 0 degrees C compared to 25 degrees C. Phosphate uptake was ATP dependent in both age groups, however, mean uptake values were significantly greater in suckling rats compared to adolescent rats. pH optimum for uptake was 7.2 p-Chloromercuribenzoate at 100 microM concentration inhibited phosphate uptake by more than 90%. Initial rate of phosphate uptake was linear up to 45 s. Kinetics of phosphate uptake at 30 s showed a Km of 0.7 +/- 0.1 and 0.15 +/- 0.1 suckling and adolescent rats, respectively. Vmax was 1.5 +/- 0.5 and 0.14 +/- 0.01 nmol/mg protein/30 s for both suckling and adolescent rats, respectively. Herein we provide evidence for the first time for the presence of a phosphate carrier in intestinal endoplasmic reticulum of rats. Endoplasmic reticulum of phosphate uptake was significantly greater in suckling rats compared to adolescent rats. This increase in uptake is due to a greater number and activity of phosphate carriers in suckling rats.
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PMID:Phosphate transport by intestinal endoplasmic reticulum during maturation. 339 95

Lipid composition of plasma membranes from luteal cells was examined to determine whether changes in this organelle occur during regression and maintenance of the corpus luteum in nonpregnant (NP) and pregnant (P) ewes, respectively. Forty ewes were assigned to be killed on Day 13 or 15 of the estrous cycle (D13-NP and D15-NP) or pregnancy (D13-P and D15-P). Purification of luteal plasma membranes on discontinuous sucrose gradients yielded two fractions, designated F1 and F2, that exhibited the greatest enrichment of 5'-nucleotidase activity (five- and fourfold, respectively) over that of the homogenate. These fractions also yielded the lowest contamination by endoplasmic reticulum as represented by nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome C reductase activity and mitochondrial membranes as indicated by succinate dehydrogenase activity. Predominant phospholipids identified in membranes obtained from all groups were phosphatidylcholine (PC, 48.9 +/- 0.6% of total phospholipid), phosphatidylethanolamine (PE, 33.3 +/- 0.4%), sphingomyelin (SPH, 9.7 +/- 0.3%), phosphatidylserine (PS, 3.5 +/- 0.2%), and phosphatidylinositol (PI, 4.0 +/- 0.5%). No changes in microgram phospholipid/mg membrane protein were observed for any luteal phospholipid on D13 and 15 of the estrous cycle or pregnancy. No significant changes in the relative percentages of major fatty acids present in PC (palmitic [16:0], oleic [18:1]), PE (stearic [18:0], 18:1 and arachidonic [20:4]), or PS (18:0, 18:1, docosatetraenoic [22:4]), nor in the ratios of unsaturated (U) to saturated (S) fatty acids in these phospholipids were observed. Significant differences in unsaturated fatty acids of chain length greater than 20 carbons present in minor quantities in PC, PE, and PS were detected between NP and P ewes as well as between days within reproductive stage. The profile of major fatty acids present in PI revealed decreases in 18:0 and 20:4 in D15-NP and increases in 22:4 and docosapentaenoic acid (22:5) in luteal membranes of both D13- and D15-NP ewes relative to the levels of these fatty acids in PI of corresponding groups of pregnant ewes. There was a general trend for 20:4 levels of PC and PI in membranes of D15-NP ewes to be inversely related to those of D15-P ewes. Collectively, these changes were reflected by an increased U:S fatty acid ratio in luteal membrane PI during the estrous cycle. Specific binding of [125I] iodo-human chorionic gonadotropin to luteal plasma membranes from NP and P ewes on D13 and 15 (6/group) revealed similar affinities and concentrations of unoccupied luteinizing hormone (LH) receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of plasma membrane lipids and luteinizing hormone receptors of ovine corpora lutea during luteolysis and early pregnancy. 340 35

Membranes purified from castor bean endosperm glyoxysomes by washing with sodium carbonate exhibited integral NADH:ferricyanide and NADH:cytochrome c reductase activities. The enzyme activities could not be attributed to contamination by other endomembranes. Purified endoplasmic reticulum membranes also contained the redox activities; and marker enzyme analysis indicated minimum cross contamination between glyoxysomal and endoplasmic reticulum fractions. The glyoxysomal redox activities were optimally solubilized at detergent to protein ratios (weight to weight) of 10 (Triton X-100), 50 (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), and 100 (octylglucoside). Detergent in excess of the solubilization optimum was stimulatory to NADH:ferricyanide reductase and inhibitory to NADH:cytochrome c reductase. Endoplasmic reticulum redox activity solubilization profiles were similar to those obtained for glyoxysomal enzymes using Triton X-100. Purification of the glyoxysomal and endoplasmic reticulum NADH:ferricyanide reductases was accomplished using dye-ligand affinity chromatography on Cibacron blue 3GA agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of NADH:ferricyanide reductase preparations purified by rate-zonal density gradient centrifugation, affinity chromatography, and nondenaturing electrophoresis of detergent-solubilized glyoxysomal and endoplasmic reticulum membranes consistently displayed 32- and 33-kDa silver-stained polypeptide bands, respectively.
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PMID:Characterization of membrane-bound electron transport enzymes from castor bean glyoxysomes and endoplasmic reticulum. 341 45

Microsomal preparations from normal human skin fibroblasts were used to investigate the role of free cholesterol in the endoplasmic reticulum in the control of cholesterol biosynthesis by regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (NADPH) [(S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]. Controlled changes in the cholesterol/phospholipid ratio of microsomes were introduced either in intact cells by incubating fibroblast cultures with whole serum or lipoprotein-deficient serum or by enrichment or depletion of cholesterol in microsomal preparations by incubating microsomes with cholesterol-rich or cholesterol-poor egg phosphatidylcholine unilamellar vesicles. Cholesterol enrichment resulted in suppression of reductase activity and increased ESR order parameters for 12-nitroxystearate in the microsomal preparations. Conversely, cholesterol depletion caused an activation of reductase and a decrease in order parameter. Enrichment of microsomal preparations with a "nonfluid" lipid, dipalmitoyl phosphatidylcholine, also suppressed enzyme activity and increased membrane order. The effect was reversed by subsequent enrichment of the microsomes with fluid egg phosphatidylcholine. Our findings suggest that cholesterol may regulate its own biosynthesis, at least in part, by suppression of hydroxymethylglutaryl-CoA reductase mediated through changes in membrane fluidity as measured by ESR order parameter.
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PMID:Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase by changes in microsomal cholesterol content or phospholipid composition. 346 43

The present study demonstrates unequivocally the existence of short-chain trans-2-enoyl coenzyme A (CoA) hydratase and beta-ketoacyl CoA reductase activities in the endoplasmic reticulum of rat liver. Subcellular fractionation indicated that all four fractions, namely, mitochondrial, peroxisomal, microsomal, and cytosolic contained significant hydratase activity when crotonyl CoA was employed as the substrate. In the untreated rat, based on marker enzymes and heat treatment, the hydratase activity, expressed as mumol/min/g liver, wet weight, in each fraction was: mitochondria, 684; peroxisomes, 108; microsomes, 36; and cytosol, 60. Following di-(2-ethylhexyl)phthalate (DEHP) treatment (2% (v/w) for 8 days), there was only a 20% increase in mitochondrial activity; in contrast, peroxisomal hydratase activity was stimulated 33-fold, while microsomal and cytosolic activities were enhanced 58- and 14-fold respectively. A portion of the cytosolic hydratase activity can be attributed to the component of the fatty acid synthase complex. Although more than 70% of the total hydratase activity was associated with the mitochondrial fraction in the untreated rat, DEHP treatment markedly altered this pattern; only 11% of the total hydratase activity was present in the mitochondrial fraction, while 49 and 29% resided in the peroxisomal and microsomal fractions, respectively. In addition, all four subcellular fractions contained the short-chain NADH-specific beta-ketoacyl CoA (acetoacetyl CoA) reductase activity. Again, in the untreated animal, reductase activity was predominant in the mitochondrial fraction; following DEHP treatment, there was marked stimulation in the peroxisomal, microsomal, and cytosolic fractions, while the activity in the mitochondrial fraction increased by only 39%. Hence, it can be concluded that both reductase and hydratase activities exist in the endoplasmic reticulum in addition to mitochondria, peroxisomes, and soluble cytoplasm.
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PMID:Hepatic subcellular distribution of short-chain beta-ketoacyl coenzyme A reductase and trans-2-enoyl coenzyme A hydratase: 25- to 50-fold stimulation of microsomal activities by the peroxisome proliferator, di-(2-ethylhexyl)phthalate. 351 53

The effects of a new piperazine derivative BM 15766, which inhibits the biosynthesis of cholesterol at the 7-dehydrocholesterol-delta 7-reductase step, upon the ultrastructure of rat liver and the serum lipids, have been investigated. Treated animals showed a marked reduction in total sterol content in serum with simultaneous reduction of triglycerides. The catalase activity in liver homogenates was unchanged, carnitine acetyltransferase increased only slightly, and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase was augmented by a factor of 2. In sections stained with alkaline 3,3'-diaminobenzidine for catalase, distinct proliferation of peroxisomes (PO) in perivenous regions of the hepatic lobules was noted in rats of both sexes. In male animals many PO showed loop-like extensions and invaginations of their limiting membranes into the matrix. Such alterations were seen less frequently in female animals; instead, females exhibited in the same regions of the hepatic lobules, large aggregates of PO, smooth endoplasmic reticulum, and mitochondria with longitudinal cristae. Close contacts of PO and fenestrated segments of smooth endoplasmic reticulum were noted in both sexes. These observations demonstrate the marked adaptive response of rat hepatocyte organelles to severe hypocholesterolemia induced by BM 15766. The alterations of PO may reflect attempts to increase their surface membrane, which plays a crucial role in the exchange of substrates between the cytoplasm and the peroxisomal matrix. Moreover, the close association of PO and smooth endoplasmic reticulum could facilitate the shuttle of lipid intermediates between these two intracellular compartments involved in the biosynthesis of complex lipids.
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PMID:Proliferation of peroxisomes in pericentral hepatocytes of rat liver after administration of a new hypocholesterolemic agent (BM 15766). Sex-dependent ultrastructural differences. 357 22

A system for the assay of 3-hydroxy-3-methyglutaryl (HMG) coenzyme A (CoA) reductase in digitonin-permeabilized Chinese hamster ovary cells is described. Under these conditions, HMG-CoA reductase remained intact and associated with the endoplasmic reticulum, and values for Km (HMG-CoA), Ki (mevinolin), and active/total activity were similar to those seen in sonicated cell preparations. However, the mechanism by which this rapidly turned over (half-life approximately 2 h) enzyme is degraded was disrupted. Addition of ATP at physiological concentrations to digitonin-permeabilized cells resulted in the rapid, irreversible loss of enzyme activity. Immunoblot analysis showed that this loss of activity was followed by cleavage of the intact 97-kilodalton enzyme to a 68-kilodalton fragment which was distinct from the catalytically active fragments generated by nonspecific proteolysis in sonicated cell homogenates. Assay of a lysosomal marker enzyme confirmed that ATP-mediated inactivation and cleavage of reductase was not due to release of lysosomal proteases. The possible role of ATP in phosphorylation, inactivation, and degradation of reductase is discussed.
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PMID:ATP-dependent degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in permeabilized cells. 358 46

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic NH2-terminal domain that contains seven apparent membrane-spanning regions and a single N-linked carbohydrate chain. The catalytic domain, which includes the COOH-terminal two-thirds of the protein, extends into the cytoplasm. The enzyme is normally degraded with a rapid half-life (2 h), but when cells are depleted of cholesterol, its half-life is prolonged to 11 h. Addition of sterols accelerates degradation by fivefold. To explore the requirements for regulated degradation, we prepared expressible reductase cDNAs from which we either deleted two contiguous membrane-spanning regions (numbers 4 and 5) or abolished the single site for N-linked glycosylation. When expressed in hamster cells after transfection, both enzymes retained catalytic activity. The deletion-bearing enzyme continued to be degraded with a rapid half-life in the presence of sterols, but it no longer was stabilized when sterols were depleted. The glycosylation-minus enzyme was degraded at a normal rate and was stabilized normally by sterol deprivation. When cells were induced to overexpress the deletion-bearing enzyme, they did not incorporate it into neatly arranged crystalloid ER tubules, as occurred with the normal and carbohydrate-minus enzymes. Rather, the deletion-bearing enzyme was incorporated into hypertrophied but disordered sheets of ER membrane. We conclude that the carbohydrate component of HMG CoA reductase is not required for proper subcellular localization or regulated degradation. In contrast, the native structure of the transmembrane component is required to form a normal crystalloid ER and to allow the enzyme to undergo regulated degradation by sterols.
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PMID:Partial deletion of membrane-bound domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase eliminates sterol-enhanced degradation and prevents formation of crystalloid endoplasmic reticulum. 358 46

In the testes of rats treated with cadmium acetate (7 or 20 mumoles/kg, 24 hr, s.c.), the activity of glutathione (GSH)-peroxidase was increased. At the same time, the activity of glutathione disulfide (GSSG)-reductase and the cellular GSH concentration were decreased significantly. The basal activity of peroxidase in the Leydig and the Sertoli cell populations was comparable. However, the magnitude of increases in the activities markedly differed in the two cell populations, with that of the Sertoli cells increasing to nearly 450% of the control value in response to treatment with 20 mumoles/kg Cd2+. In the Leydig cells, the enzyme activity in response to the same treatment increased to only about 170% of the control value. Cd2+ treatment increased the concentration of heme in the microsomal and the smooth and rough endoplasmic reticulum fractions of the whole testis, as well as in the microsomal fractions of the Leydig and the Sertoli cells. As with the peroxidase activity, the two cell populations vastly differed in their susceptibilities to Cd2+ treatment, with the Sertoli cells being more severely affected by the metal. In the Sertoli cells the microsomal heme concentration was increased by approximately 11-fold, whereas only a 2-fold increase in the Leydig cells was noted. The increase in GSH-peroxidase activity was not due to the peroxidase activity of GSH-S-transferases, insofar as an increase in transferase activity was not observed in the Leydig and the Sertoli cells. Treatment of rats with sodium selenite (10 mumoles/kg, s.c.) 30 min before Cd2+ treatment (20 mumoles/kg) fully suppressed the above-described spectrum of effects of Cd2+ in the testis. Also, sodium selenite at a lower dose of 5 mumoles/kg prevented an increase in GSH-peroxidase activity. It is hypothesized that increased GSH-peroxidase activity in the Leydig and the Sertoli cells constitutes an adaptive response to increased cellular levels of heme and to the free radicals generated by the heme molecule. Selenium prevents the increase in GSH-peroxidase activity by circumventing the increase in cellular heme concentration. The protection is believed to be related, at least in part, to increased production of cellular GSH.
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PMID:Differential effect of cadmium on GSH-peroxidase activity in the Leydig and the Sertoli cells of rat testis. Suppression by selenium and the possible relationship to heme concentration. 359 23

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