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)

Injection of leupeptin (an inhibitor of lysosomal cathepsins B, H, and L) to nonstarved rats causes an expansion of the autophagic vacuolar (AV) system in hepatocytes. Readily identifiable cytoplasmic constituents were seen within the AVs shortly after the administration. Later, the contents of the AVs seemed to reach more advanced stages of degradation. Liver AVs were purified by a one-step centrifugation of a crude mitochondrial lysosomal fraction in a discontinuous metrizamide gradient after exposing the rats to leupeptin for varying periods of time. Leupeptin caused alterations of the AV fraction that were time dependent. Initially, i.e., after 30 minutes of leupeptin exposure, mature (secondary) lysosomes clearly dominated over nascent AVs. The situation was reversed when fractionation was performed 1 or 2 hours following the injection of leupeptin. Now, the AVs were more frequent than the mature lysosomes. Later, the proportion of mature lysosomes was again larger. An increase in dense bodies was noted after 16 hours of leupeptin treatment. The proteolytic capacity of the AVs at different stages of maturation was measured after labeling liver proteins with an injection of L-1-14C-leucine 16 hours before sacrifice. AVs were purified after varying times of exposure to leupeptin. The proteolysis decreased greatly 1 to 2 hours following the injection of leupeptin but never ceased. On the other hand, lipolysis seemed unaffected by leupeptin using a similar experimental protocol as for proteolysis. If the animals were subjected to more lasting exposure to leupeptin before fractionation, proteolysis increased, displaying a peak higher than control, occurring after approximately 4 hours. The degradation gradually returned to control values after 16 hours. A catch-up in proteolysis was thus observed. The time course of proteolysis was reflected in the protein content in the AV fraction. After an initial increase that coincided with the lowered proteolysis, it returned to control level. Marker enzyme activities for endoplasmic reticulum and mitochondria (G6Pase and succinate-cytochrome c reductase) followed the same pattern. The AV content of the cytosolic enzymes lactate dehydrogenase and aldolase reached as high as 2.30 and 2.80% of the values in the homogenate during the 1st hour of leupeptin exposure. From these data the half-lives of the enzymes were calculated. They were: for aldolase, 43 hours; for LDH, 68 hours. This suggests that AVs account for a substantial proportion of degradation not only of organelles but also of soluble cytosolic enzymes.
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PMID:Effect of leupeptin on the autophagic vacuolar system of rat hepatocytes. Correlation between ultrastructure and degradation of membrane and cytosolic proteins. 633 Apr 53

We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983, Nature (London) 306:67-69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000 X g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of Ip3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r = 0.96, 1.00, 0.91, respectively) and NADPH cytochrome c reductase (r = 0.63, 0.98, 0.90, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochrome c oxidase (r = -0.64) and glutamate dehydrogenase (r = -0.75) and with the plasma membrane markers (Na+ + K+)-ATPase (r = -0.81) and alkaline phosphatase (r = -0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.
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PMID:Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas. 633 62

Previous immunolabeling studies (Roman, L.M., and A.L. Hubbard, 1983, J. Cell Biol., 96:1548-1558; Roman, L.M., and A.L. Hubbard, 1984, J. Cell Biol., 98:1488-1496, companion paper) established leucine aminopeptidase (LAP) as a specific marker for the bile canalicular (BC) domain of the rat hepatocyte plasma membrane (PM). In this study, we have isolated membrane from a sonicated PM vesicle fraction using anti-LAP-coated Staphylococcus aureus cells as a solid-phase immunoadsorbent. The extent and specificity of the immunoadsorption were assessed by following the behavior of LAP (the BC marker) and 32P-labeled membrane phospholipids (a uniform membrane marker). The BC fraction obtained was significantly enriched in LAP (yield: greater than 70% of PM-LAP). Alkaline phosphatase, 5'-nucleotidase, and a 110,000-dalton glycoprotein, HA-4, were enriched in the BC fraction to the same extent as LAP (enzyme or antigen/LAP = 1.0). However, alkaline phosphodiesterase I was not enriched to the same degree (enzyme/LAP = 0.5). Contamination of this BC fraction by membrane derived from the sinusoidal domain and endoplasmic reticulum, as determined from the distribution of the asialoglycoprotein receptor and NADH cytochrome c reductase, respectively, was small (less than 13%).
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PMID:A domain-specific marker for the hepatocyte plasma membrane. III. Isolation of bile canalicular membrane by immunoadsorption. 637 Oct 22

The ingestion of ethanol results in altered compositions of the polyenoic fatty acids in a variety of liver and brain membranes. A possible cause for these alterations in hepatic endoplasmic reticulum membranes has been studied by measuring the delta 9, delta 6, and delta 5 acyl-CoA desaturase activities in hepatic microsomes from chronically or acutely treated rats. Chronically, ethanol decreased all three enzyme activities with the following order of sensitivity: delta 6 (65%), delta 9 (54%), and delta 5 (46%). The short-term study indicated that all three desaturase activities were affected after 1 day of ethanol feeding. NADPH- and NADH-cytochrome c reductase activities were found to be reduced in chronically treated rats, and the NADH-cytochrome c reductase was decreased in the acutely treated. However, these reduced enzyme activities could not account for the decrease in desaturase activities due to the very marked differences between the specific activities of these enzymes compared to the desaturase. Thus, we conclude that changes in membrane polyenoic fatty acid composition can be result of ethanol-induced decreases in the terminal desaturase enzymes.
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PMID:Ethanol ingestion and polyunsaturated fatty acids: effects on the acyl-CoA desaturases. 640 39

ATP promotes 45Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca2+-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the 45Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5'-nucleotidase. In contrast, the additional Ca2+-transport activity elicited by oxalate behaved like NADH-cytochrome c reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca2+-storage capacity was not detectable in the absence of Ca2+-trapping agent. Low digitonin concentrations selectively increased the Ca2+ permeability of the plasmalemmal vesicles. The two Ca2+-transport activities were further differentiated by their distinct sensitivity of K+, vanadate and calmodulin. In this respect, the oxalate-insensitive and oxalate-stimulated Ca2+-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca2+ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.
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PMID:Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle. 645 27

Lymph node cell homogenates were fractionated by differential or isopycnic centrifugation and the fractions analyzed for biochemical markers with particular focus on plasma membrane constituents. Markers for the nucleus (DNA), mitochondria (cytochrome oxidase), and lysosomes (acid hydrolases) showed the expected distributions which were different from those of membrane-bound enzymes. 5'-Nucleotidase, alkaline phosphodiesterase, gamma-glutamyltranspeptidase, and cholesterol were membrane-bound and distributed identically after isopycnic centrifugation with peaks at 1.15. The distributions of the enzymes were all shifted to higher densities by digitonin treatment, confirming their association with plasma membrane-derived elements. The distribution of galactosyltransferase (ovalbumin acceptor), largely overlapped those of plasma membrane markers but it was only slightly shifted by digitonin, suggesting its localization in Golgi apparatus. The distribution of mannosyltransferase (dolichyl phosphate acceptor) also overlapped those of plasma membrane and Golgi markers but it was centered at higher density (1.18) and was unaffected by digitonin. It is a useful marker for endoplasmic reticulum. 50% of the activity was in low speed "nuclear" sediments where it was associated with the nuclear membrane. A number of other putative and previously used markers for the endoplasmic reticulum of lymphocytes were shown not to be localized in these membranes. In particular, NADH-cytochrome c reductase was only partly associated with the endoplasmic reticulum (56%) and the remainder of the activity was in mitochondria (44%). The results show the heterogeneity in equilibrium density of plasma membrane vesicles and the considerable overlap of their distribution with those of other cellular membranes; they should provide a basis for the more rational design of preparative procedures for the lymphocyte plasma membrane.
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PMID:Characterization of rat lymphocyte cell membranes by analytical isopycnic centrifugation. 660 29

S-Adenosylhomocysteine (AdoHcy) binding to various membrane fractions of rat liver was determined at pH 7.4, using an oil centrifugation technique. The highest binding activity was found in the heavy microsomal (M-H) fraction enriched in endoplasmic reticulum, but high binding activity was also observed in the light microsomal fractions enriched in blood sinusoidal membranes (M-L fraction), and the heavy nuclear fraction (N-H fraction) containing the contiguous area. A substantial portion of AdoHcy binding activity in the M-L fraction may be ascribed to contamination of this fraction with endoplasmic reticulum, as indicated by the distribution of NADPH cytochrome c reductase activity. Binding activity was low in the light nuclear (N-L) fraction corresponding to the bile canaliculi. Phospholipid methyltransferase activity was determined in the same membrane fractions under similar conditions (pH 7.4), and in the absence and presence of added phospholipids. The distribution of the enzyme activity was dependent on the presence of exogenous phospholipids, and grossly similar to AdoHcy binding, the highest activities being observed in the M-H and the M-L fractions. The N-H fraction, rich in AdoHcy-binding activity, demonstrated, however, a very low phospholipid methyltransferase activity. It is concluded that AdoHcy-binding activity is not confined to the plasma membranes, and a major fraction of the binding activity resides on membranes derived from the endoplasmic reticulum. Also, the present results add to previous data suggesting that phospholipid methyltransferase does not totally account for the AdoHcy-binding sites on rat liver membranes.
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PMID:Binding of S-adenosylhomocysteine to various domains of the plasma membrane and to the endoplasmic reticulum from rat liver: relation between binding and phospholipid methyltransferase activity. 666 23

The usual histologic pattern in acute viral hepatitis (AVH) includes cellular abnormalities predominantly in the perivenular (zone 3) hepatocytes and changes interpreted as representing regenerative activity in the periportal (zone 1) hepatocytes. Enzyme histochemical and ultrastructural studies of livers of 12 patients with AVH were undertaken to see whether these features support the concept of regeneration of hepatocytes in zone 1. The swollen hepatocytes in the perivenular areas were hydropic, with dilated or eccentric rough endoplasmic reticulum and decreased or vesicular smooth endoplasmic reticulum; correspondingly, the glucose-6-phosphatase activity (reflecting, when present, intact and functional endoplasmic reticulum) was markedly decreased. Succinic dehydrogenase and diphosphopyridine nucleotide diaphorase activities, representing mitochondrial enzymes, were limited to the perinuclear or pericanalicular cytoplasm of swollen hepatocytes. gamma-Glutamyl transpeptidase activity was increased. The periportal hydropic hepatocytes were small and arranged in clusters displacing sinusoids. Ultrastructurally, these hepatocytes had nearly normal organelles but scanty smooth endoplasmic reticulum. Activities of the enzymes glucose-6-phosphatase, succinic dehydrogenase, and diphosphopyridine nucleotide diaphorase were weak, although glycogen was abundant. gamma-Glutamyl transpeptidase activity was scanty in these hepatocytes. These findings from enzyme histochemical and electron microscopic studies could be interpreted as evidence of functional deterioration of perivenular swollen hepatocytes and relative functional immaturity of periportal hydropic clustered hepatocytes, suggesting regeneration of zone 1 hepatocytes.
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PMID:Acute viral hepatitis: morphologic and functional correlations in human livers. 669 43

Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2-3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose-6-phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome c reductase and contain membrane vesicles with attached ribosomes. K+-stimulated p-nitrophenyl phosphatase and (Na+K+) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.
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PMID:Isolation of plasma membrane, golgi apparatus, and endoplasmic reticulum fractions from single homogenates of mouse liver. 670 2

The effects of seasonal feeding on the structure and function of the hepatic microsomes of the reindeer were studied. In native microsomes the protein content did not vary depending on seasonal feeding, but there was a slight modification of the lipid moiety. The sensitivity of microsomes to membrane treatments changed from season to season. Seasonal feeding did not affect the hepatic cytochrome c reductase activity. However, the poor winter food depressed UDPglucuronosyltransferase activity to one third of that after summer feeding. The UDPglucuronosyltransferase activity was lower in both native and treated microsomes. No differences were found in serum activities of aspartate or alanine aminotransferase although there were changes in the structure and function of hepatocytes. The poor winter feeding of the reindeer decreases their capacity to glucuronate foreign compounds and this is mediated by structural modification on the hepatic endoplasmic reticulum.
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PMID:On the effects of nutrition on the metabolism of foreign compounds in the liver of reindeer (Rangifer tarandus tarandus L). 676 22


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