Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of the study was to consider quantitatively the relationships between the surface area of the endoplasmic reticulum (ER) and constituent marker enzyme activities, as they occur in fractions collected from rat liver homogenates. The ER surface area was estimated in five membrane-containing fractions by use of a combined cytochemical-stereological technique (5), while, at the same time, ER marker enzymes were assayed biochemically. Fraction/homogenate recoveries for the ER enzymes averaged 100%, total membrane surface area 98%, and ER surface area 96%. Relative specific activities, which compare the relative amounts of ER marker enzyme activities to the relative ER surface area in the membrane-containing fractions, indicate variable distributions for glucose-6-phosphatase and NADPH cytochrome c reductase, but not for esterase.
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PMID:Integrated stereological and biochemical studies on hepatocytic membranes. I.V. Heterogeneous distribution of marker enzymes on endoplasmic reticulum membranes in fractions. 624 65

Endoplasmic reticulum membrane-rich fraction was obtained by subfractionation of the light microsomes from mouse jejunal mucosal epithelial cells. It was marked by high glucose-6-phosphatase, NADPH-cytochrome c reductase, and NADH-cytochrome c reductase activities and low Na+,K+-ATPase activity. The enrichment of Na+,K+-ATPase was 180-fold higher in the basolateral membranes than in the endoplasmic reticulum membrane-rich fraction relative to glucose-6-phosphatase. The protein peak that was phosphorylated in a Na-dependent manner was prominent in the basolateral membranes while it was a minor peak in the endoplasmic reticulum membrane-rich fraction. Under the electron microscope the fraction was seen to be composed of homogeneous small vesicles with thin smooth membranes.
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PMID:Endoplasmic reticulum membrane isolated from small-intestinal epithelial cells: enzyme and protein components. 627 65

1. Plasma membranes were isolated from Krebs II ascite cells grown in the mouse. Cells were disrupted by nitrogen cavitation in an isotonic alkaline buffer containing magnesium and ATP. Isolation was performed in an alkaline-buffered self-generating gradient of Percoll with an angular rotor. At each step of the preparation, the pH appeared as the critical aspect of our procedure. 2. External membrane markers were concanavalin A and 5'-nucleotidase (EC 3.1.3.5). They reached a relative specific activity of 10, whereas this value was only of 0.7 for the endoplasmic reticulum marker, NADH dehydrogenase (EC 1.6.99.3). 3. Plasma membrane from 4 ml packed cells were isolated within 1 h after homogenization with good yield: 50% and 67% of total [3H]concanavalin A and 5'-nucleotidase, respectively, were recovered in the two plasma membrane fractions. 4. Electron microscopy examination showed the presence of vesicles of different sizes devoid of other structural contaminants. 5. Using the specific binding of concanavalin A to the external cell membrane, it was calculated that about 50% of the total cell phospholipid and 10% protein are located in the plasma membrane. Their sphingomyelin content is much higher than in the whole cell, in contrast to phosphatidylinositol, known as a more specific endoplasmic reticulum phospholipid.
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PMID:Isolation and characterization of plasma membranes from krebs II ascite cells using Percoll gradient. 628 35

Resident peritoneal macrophages of the mouse, cultivated for 3 d, have been studied by quantitative subcellular fractionation using differential centrifugation and density equilibration in linear gradients of sucrose. Density equilibration experiments were carried out on untreated cytoplasmic extracts, on cytoplasmic extracts treated with digitonin or sodium pyrophosphate, and on cytoplasmic extracts derived from cells cultivated for 24 h in the presence of Triton WR-1339. The enzyme distributions obtained distinguished six typical behaviors characteristic of distinct subcellular entities. Acid alpha-galactosidase and other acid hydrolases displayed the highest average velocity of sedimentation and equilibrium density. Culturing in a medium that contained Triton WR-1339 markedly decreased their density, most likely as a result of Triton WR-1339 accumulation within lysosomes. Cytochrome c oxidase and the sedimentable activity of malate dehydrogenase showed a narrow density distribution centered around 1.17, very similar under all the experimental situations; their rate of sedimentation fell within the range expected for mitochondria. Catalase was particle-bound and exhibited structure-linked latency (80 percent); it was released in soluble and fully active form by digitonin, but this required a much higher concentration than in the case of lysosomal enzymes. Differences relative to all the other enzymes studied suggest the existence of a particular species of organelles, distinctly smaller than mitochondria, and possibly related to peroxisomes. Many enzymes were microsomal in the sense that the specific activities, but not the yields, were greater in microsomes than in other fractions obtained by differential centrifugation. These enzymes were distinguished in three groups by their properties in density equilibration experiments. NAD glycohydrolase, alkaline phosphodiesterase I, and 5'-nucleotidase had low equilibrium densities but became noticeably more dense after addition of digitonin. The other microsomal enzymes were not shifted by digitonin, in particular N-acetylglucosaminyltransferase and galactosyltransferase, which otherwise equilibrated at the same position in the gradient. We assign the digitonin-sensitive enzymes to plasma membranes and possibly to related endomembranes of the cells, and the two glycosyltransferases to elements derived from the Golgi apparatus. Finally, alpha-glucosidase, sulphatase C, NADH cytochrome c reductase, NADPH cytochrome c reductase, and mannosyltransferase, equilibrated at a relatively high density but were shifted to lower density values after addition of sodium pyrophosphate. These properties support their association with elements derived from the endoplasmic reticulum.
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PMID:Analytical subcellular fractionation of cultivated mouse resident peritoneal macrophages. 630 Feb 79

A method has been devised for the isolation of a highly purified preparation of lamellar bodies from rabbit lung. The purity of the preparation was confirmed by electron microscopy, marker enzymes, phospholipid composition, and isopycnic centrifugation on continuous density sucrose gradients. Contamination of the lamellar bodies by such subcellular components as mitochondria, nuclei, lysosomes and plasma membranes could be excluded; however, reduced nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome c reductase, an enzyme specific for the endoplasmic reticulum components was a persistent contaminant in the preparation of the isolated lamellar bodies. When the lamellar bodies were subject to isopycnic centrifugation, all of the NADPH cytochrome c reductase activity was associated with the lamellar bodies in the low density peak; no reductase activity could be detected in the region of the density gradient demonstrated to localize microsomes. Use of 3H-radiolabeled microsomes confirmed that all of the NADPH cytochrome c reductase activity present in the lamellar body preparations could be accounted for by microsomal contamination. When lamellar bodies or liposomal membranes synthesized from the total phospholipid fraction of lamellar bodies were analyzed by the electron paramagnetic resonance probe, 5-dioxyl-methylstearate, they exhibited a high degree of fluidity at physiological temperature. This was in contrast to the low fluidity of liposomal membranes composed of pure dipalmitoylphosphatidylcholine, the major component (50%) of rabbit lamellar body phospholipids. Furthermore, the major temperature-dependent phase transition in lamellar body membranes occurred at a different temperature (30.5 degrees C) from that of dipalmitoyl-phosphatidylcholine (41.0 degrees C). It is clear, therefore, that the membrane fluidity of lamellar bodies must be highly influenced by the minor lipid component.
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PMID:Isolation and characterization of rabbit lung lamellar bodies. 630 68

The endoplasmic reticulum from Neurospora crassa was identified by monitoring the activity of the putative enzyme marker phosphatidylcholine glyceride transferase. After differential centrifugation of a cell homogenate, phosphatidylcholine glyceride transferase activity initially copurified with plasma membrane H+-ATPase. However, isopycnic centrifugation of the whole-cell homogenate on a linear sucrose gradient separated the two enzyme activities into different fractions. The lighter membrane fraction exhibited characteristics that have been associated with the endoplasmic reticulum in other organisms: (i) the inclusion of magnesium caused this light membrane fraction to shift to a higher density on the gradient; (ii) it was highly enriched in cytochrome c reductase, an endoplasmic reticulum marker in other systems; and (iii) the morphology of the light fraction with and without added magnesium was clearly distinguishable from that of the plasma membrane fraction by electron microscopy. A reinvestigation of the location of chitin synthetase confirmed its association with the plasma membrane fraction even after separation of the lighter fractions.
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PMID:Isolation and characterization of the Neurospora crassa endoplasmic reticulum. 631

The reduction and the potential autoxidation of quinoid compounds may be viewed as taking place in three cell compartments. In microsomal fractions (endoplasmic reticulum) one-electron reduction by NAPDH-cytochrome P450 reductase leads to the formation of semiquinones which rapidly react with oxygen to form the parent quinone and superoxide anions. The formation of superoxide through this futile cycle leads ultimately to other damaging species (H2O2 and .OH). A similar futile cycle in mitochondria involves NADH dehydrogenase. In this instance, mitochondria initiation of such a cycle with quinones results not only in the formation of toxic radical species but also in the diversion of electrons from phosphorylating pathways. The consequent diminution of cellular ATP may have as important a consequence with respect to the toxicity of quinones as the generation of radicals. Finally, cytosolic DT diaphorase, which carries out a two-electron reduction of quinones to more stable hydroquinones, may compete with the one-electron systems and participate in the detoxification of quinones by supplying hydroquinones for conjugation reactions. The extent of quinone-induced damage may thus vary from cell to cell depending on the integration of these pathways.
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PMID:Futile redox cycling: implications for oxygen radical toxicity. 631 61

Distribution of specific binding sites for [3H]nitrendipine was studied in subcellular fractions isolated from rat gastric fundus smooth muscle and from rat myometrium. There was an excellent correlation between the distribution of [3H]nitrendipine binding determined at the nitrendipine concentrations of 0.138 and 1.38 nM, and the distribution of the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase, phosphodiesterase I, and Mg-ATPase, but not between the mitochondrial markers cytochrome c, oxidase, succinate-dependent cytochrome c reductase, or rotenone-insensitive NADH-dependent cytochrome c reductase or the putative endoplasmic reticulum marker NADPH-dependent cytochrome c reductase. The binding occurred with high affinity and with a similar (0.097-0.146 nM) equilibrium dissociation constant to all the fractions, even though the density of binding sites varied and was highest in the plasma membrane marker-enriched fractions. The maximal binding in the plasma membrane-enriched fraction from the rat gastric fundus smooth muscle was 0.43 +/- 0.04 pmol/mg, and in that from rat myometrium was 0.72 +/- 0.09 pmol/mg. Thus in the two smooth muscles studied the plasma membrane is the locus of the high affinity nitrendipine binding.
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PMID:Subcellular distribution of [3H]nitrendipine binding in smooth muscle. 632 63

NAD kinase activity from dark grown corn coleoptiles is shown to be almost totally dependent on Ca2+ and calmodulin. Nearly all of the enzyme activity is found in a particulate fraction. Upon differential and density gradient centrifugation the NAD kinase activity co-migrates with the mitochondrial cytochrome c oxidase whereas marker activities for nuclei, etioplasts, endoplasmic reticulum, and microbodies could well be separated, indicating that the NAD kinase is associated with mitochondria. This NAD kinase, associated with intact mitochondria, can be activated by exogenously added Ca2+ and calmodulin. In order to investigate the submitochondrial localization of the NAD kinase, the organelles were ruptured by osmotic treatment and sonication and the submitochondrial fractions were separated by density gradient centrifugation. The NAD kinase activity exhibits the same density pattern as the antimycin A-insensitive NADH-dependent cytochrome c reductase, a marker enzyme of the outer mitochondrial membrane. Marker enzymes for the mitochondrial matrix and the inner mitochondrial membrane reveal different density profiles. These results indicate that the Ca2+, calmodulin-dependent NAD kinase from coleoptiles of dark grown corn seedlings is located at the outer mitochondrial membrane. The physiological relevance of the location and the Ca2+, calmodulin-dependence of the NAD kinase will be discussed.
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PMID:A Ca2+, Calmodulin-dependent NAD kinase from corn is located in the outer mitochondrial membrane. 632

Luteal gonadotropin receptors decrease in cows, sheep and rats within 24 h following an injection of a luteolytic dose of prostaglandin (PG) F2 alpha. But it is not known whether this decrease is the specific event, or a reflection of general decline in luteal cell structure, function and metabolism. In order to investigate this possibility, 15 of 21 heifers were given on day 9 of the estrous cycle, a single 500 micrograms injection of Cloprostenol (CO), a synthetic PGF2 alpha analog. These heifers were ovariectomized in groups of 5 at 12, 24 and 36 h after CO. For controls, a group of 6 heifers were ovariectomized just prior to injection of the others. Serum progesterone levels decreased whereas LH levels increased (P less than 0.05) by 12 h with no additional changes observed at 24 or 36 h. The luteal plasma membranes [125I]hCG specific binding, as well as 5'-nucleotidase (5'-NE) activity, decreased by 12 h and continued to decline (P less than 0.05) until 24 h (binding) or 36 h (5'-NE). Scatchard analysis showed that the decrease in [125I]hCG binding was due to a decrease in receptor number rather than a decrease in receptor affinity. The activities of cytochrome c oxidase in mitochondria, NADH cytochrome c reductase in rough endoplasmic reticulum and galactosyl transferase in Golgi decreased while NAD pyrophosphorylase in nuclei virtually disappeared following the injection of CO. The beta-N-acetyl-D-glucosaminidase (a lysosomal hydrolase) activity in the homogenate increased by 12 h and continued to increase up to 36 h.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Decrease of various luteal enzyme activities during prostaglandin F2 alpha-induced luteal regression in bovine. 632 72


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