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)

Homogenates of cultured rat embryo fibroblasts have been assayed for acid phosphatase, N-acetyl-beta-glucosaminidase, cathepsin D, acid deoxyribonuclease, cytochrome oxidase, NADH cytochrome c reductase, 5'-nucleotidase, inosine diphosphatase, acid pyrophosphatase, neutral pyrophosphatase, esterase, catalase, cholesterol, and RNA. The validity of the assay conditions was checked. Neutral pyrophosphatase is a readily soluble enzyme. Acid hydrolases, except acid pyrophosphatase, are particle-bound enzymes, which exhibit a high degree of structural latency. They are activated and solubilized in a parallel fashion by mechanical treatments and tensio-active agents. Catalase is also particle-bound and latent; activating conditions stronger than those for hydrolases are required to activate the enzyme. Acid pyrophosphatase, 5'-nucleotidase and inosine diphosphatase are firmly particle-bound, but not latent; they are not easily solubilized. In differential and isopycnic centrifugation, the latent hydrolases, cytochrome oxidase and catalase dissociate largely from each other; this suggests the occurrence of lysosomes and peroxisome-like structures besides mitochondria. The distribution patterns of 5'-nucleotidase and cholesterol are largely similar; digitonin influences their equilibrium density to the same extent; these two constituents are thought to be related to the plasma membrane. Inosine diphosphatase and acid pyrophosphatase are also partially associated with the plasma membrane, although some part of these enzymic activities probably belongs to other structures. NADH cytochrome c reductase is associated partly with the endoplasmic reticulum, partly with mitochondria.
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PMID:Analytical fractionation of homogenates from cultured rat embryo fibroblasts. 437 90

Microsomal membranes are postulated to contain either a homogeneous arrangement of individual enzymes or groupings of functionally related enzymes. In the present study we attempt to distinguish between these hypotheses in subfractions of rough microsomes from rat liver. After sonication, the individual vesicles that make up the rough-membrane fraction average less than 1/100 of their previous mass. The vesicles in the sonicated suspension are fractionated roughly according to size on a continuous sucrose gradient. Enzyme activity or concentration in fractions of the gradient is expressed on a phospholipid basis. Fractions containing primarily small vesicles differ from those containing larger vesicles in a manner suggesting a certain degree of separation of NADH-linked from NADPH-linked enzymes. NADH-ferricyanide reductase, NADH-cytochrome c reductase and cytochrome b(5) are most concentrated within the large vesicles in the lowest third of the gradient. In contrast, NADPH-cytochrome c reductase and cytochrome P-450 are found in highest concentration in the small vesicles that make up the upper third of the gradient. The results suggest a nonrandom distribution of these two enzyme groups in the membranes of the endoplasmic reticulum.
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PMID:Heterogeneous distribution of enzymes in submicrosomal membrane fragments. 438 25

1. Induction of the formation of lipid peroxide in suspensions of liver microsomal preparations by incubation with ascorbate or NADPH, or by treatment with ionizing radiation, leads to a marked decrease of the activity of glucose 6-phosphatase. 2. The effect of peroxidation can be imitated by treating microsomal suspensions with detergents such as deoxycholate or with phospholipases. 3. The substrate, glucose 6-phosphate, protects the glucose 6-phosphatase activity of microsomal preparations against peroxidation or detergents. 4. The loss of glucose 6-phosphatase activity is not due to the formation of hydroperoxide or formation of malonaldehyde or other breakdown products of peroxidation, all of which are not toxic to the enzyme. 5. All experiments lead to the conclusion that the loss of activity of glucose 6-phosphatase resulting from peroxidation is a consequence of loss of membrane structure essential for the activity of the enzyme. 6. In addition to glucose 6-phosphatase, oxidative demethylation of aminopyrine or p-chloro-N-methylaniline, hydroxylation of aniline, NADPH oxidation and menadione-dependent NADPH oxidation are also strongly inhibited by peroxidation. However, another group of enzymes separated with the microsomal fraction, including NAD(+)/NADP(+) glycohydrolase, adenosine triphosphatase, esterase and NADH-cytochrome c reductase are not inactivated by peroxidation. This group is not readily inactivated by treatment with detergents. 7. Lipid peroxidation, by controlling membrane integrity, may exert a regulating effect on the oxidative metabolism and carbohydrate metabolism of the endoplasmic reticulum in vivo.
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PMID:Effects of lipid peroxidation on membrane-bound enzymes of the endoplasmic reticulum. 439 3

1. Marker enzymes for the mitochondrial matrix, inner membrane, inter-membrane space and outer membrane were measured in mitochondria isolated from control and regenerating rat liver. The specific activity of these enzymes was then followed for up to 30 days after operation. 2. The specific activity of marker enzymes for the matrix, inner membrane and inter-membrane space remained constant during liver regeneration. 3. However, the specific activities of monoamine oxidase and kynurenine hydroxylase, both outer-membrane markers, fell by 67% and 49% respectively from their control values at 4 days after operation, and returned to normal by about 3 weeks. 4. The repression of kynurenine hydroxylase activity was shown to be unrelated to any independent variation in tryptophan catabolism, based on tryptophan pyrrolase assays. 5. These results are considered to indicate that enzymes of the inner and outer mitochondrial membranes are synthesized asynchronously during morphogenesis. 6. The enzyme complement of purified outer membrane at 4 days after operation was about 50% of that of the appropriate control. Thus the composition of the outer membrane itself may vary dramatically, and supports the concept that constitutive enzymes may turn over independently of a membrane's existence. 7. The behaviour of the rotenone-insensitive, NADH cytochrome c reductase did not parallel the other outer-membrane enzymes for intact mitochondria, but did so when assayed in highly purified fractions of outer membrane. This suggests a labile binding to the outer membrane during the early stages of morphogenesis. 8. Electrophoresis of inner- and outer-membrane proteins revealed little difference between control and experimental mitochondria at 4 days, except for an increase in several, high-molecular-weight components of the outer membrane. These bands closely correspond to similar bands derived from smooth endoplasmic reticulum. 9. The results are discussed in relation to the biogenesis and turnover of mitochondria, and are considered to provide evidence for turnover as a unit, at least for the matrix, inner membrane, inter-membrane space and possibly some form of primary outer membrane.
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PMID:Inner- and outer-membrane enzymes of mitochondria during liver regeneration. 549 70

Small (15-18 microns) and large (18-45 microns) luteal cells were obtained from bovine corpora lutea of pregnancy by centrifugal elutriation of enzymatically dispersed luteal cells. Small luteal cells accounted for about 85% and large luteal cells for 8-12% of total luteal cell population. Small luteal cells were characterized by a low cytoplasmic/nuclear ratio with cytoplasm containing mitochondria, lysosomes, lipid droplets, dense granules and endoplasmic reticulum. Large luteal cells possessed a higher cytoplasmic/nuclear ratio with cytoplasm containing more abundant mitochondria, lipid droplets, dense granules and lysosomes compared to small luteal cells. Some of the mitochondria were very long. Both small and large luteal cells contained scarce amounts of Golgi elements. Dense granules were found close to the nucleus in both cell types. The nucleus of both cell types was acentric, irregular in shape and contained a well-defined nucleolus. The highly condensed chromatin in small luteal cells was found at the nuclear periphery and in the central region. Dispersed chromatin was found throughout the nucleus with condensed chromatin at the nuclear periphery of large luteal cells. Macrophages and fibroblasts were occasionally found in small luteal cell preparations, but their morphology was quite distinct from both small and large luteal cells. Scanning electron microscopy revealed that the majority of the small and large luteal cells were spherical or slightly elongated in shape. Small luteal cells displayed the presence of blebs, ruffles and short microvilli. Large luteal cell surface contained ruffles and randomly distributed clusters of blebs of different sizes, predominantly spherical in shape with a smooth surface. Finger-like projections were also occasionally seen. Small luteal cells contained significantly lower amounts of protein, but the ratios between protein and DNA were similar in both cell types. The basal, human chorionic gonadotropin (hCG)- or cyclic AMP-stimulated progesterone production, the apparent dissociation constants for [125I]hCG binding and the apparent total number of available sites per cell were similar in small and large luteal cells. The activities of enzymes that are involved directly or indirectly in progesterone biosynthesis and those involved in general cellular metabolism and biosynthesis were also similar in small and large luteal cells with one exception. That is, the activities of 5'-nucleotidase and NADH cytochrome c reductase were significantly higher in small compared to large luteal cells.
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PMID:Morphological and biochemical characterization of small and large bovine luteal cells during pregnancy. 608 29

A method is described for the preparation of plasma membrane enriched fractions from bovine retinal pigment epithelium (RPE) by means of differential centrifugation followed by the use of self forming gradients of Percoll. A detailed analysis of the distribution of organelle specific markers (nuclei, mitochondria, lysosomes, endoplasmic reticulum, cytosol) in the different fractions is presented. Comparison of 125I-wheat germ agglutinin (WGA) binding with more conventional plasma membrane enzyme markers demonstrates that also in RPE radiolabeled lectin is a specific and extremely sensitive marker to follow quantitatively the distribution of outer cell membranes. Results of 125I-WGA displacement experiments indicate that plasma membranes are mostly (90%) composed of right side out vesicles or sheets. On the basis of 125I-WGA radioactivity the overall recovery of plasma membranes was about 10% and purification over 15 fold. NADH cytochrome c reductase activity, which is shown to be a specific marker for endoplasmic reticulum in retinal pigment epithelium, has been utilized to evaluate microsomal contamination of the plasma membrane preparation.
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PMID:The isolation by self forming gradients of Percoll of plasma membrane enriched fractions from bovine retinal pigment epithelium. 609

The rates of heat denaturation of a protein component of endoplasmic reticulum, NADPH cytochrome c reductase, measured in the temperature range of 37-47 degrees C, show that this protein is highly unstable in the range of temperatures used in clinical hyperthermia. The rate of enzyme inactivation increases some 20-fold as the temperature increases from 37 degrees C to 45 degrees C. The enzyme has an in vitro half-life of only 7 h due to thermal inactivation at 37 degrees C. This suggests a general scheme for the physiological degradation pathway of intracellular proteins in which the first step is rapid thermal denaturation of the molecule followed by a second and slower step of proteolytic degradation. The rapid physiological turnover of intracellular proteins may be an unavoidable cost of maintaining metabolic precision in the presence of thermal noise at normal body temperature.
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PMID:Thermal instability in the endoplasmic reticulum of the rat hepatocyte. 612 36

Mg-ATP-dependent 45Ca2+ uptake and Ca2+-ATPase activity have been examined in isolated microsomes obtained by differential centrifugation and in purified subcellular fractions obtained by Ficoll-sucrose density centrifugation in the presence of mitochondrial inhibitors. Mg-ATP-dependent 45Ca2+ uptake increased with increasing EGTA-buffered free [Ca2+], reaching a maximum of 2 nmol 45Ca2+ X 15 min-1 X mg prot-1 at 2 mumol/1 [Ca2+] in the incubation medium. Half-maximal 45Ca2+ uptake was at approximately 0.2 mumol/1 [Ca2+]. Maximal Ca2+ -Mg2+ -ATPase activity was 130 nmol X 15 min-1 X mg prot-1 at 2 mumol/l [Ca2+], with an apparent Km of approximately 0.3 mumol/l [Ca2+]. The Ca2+ ionophore A23187 (10(-6) mol/l), the mercurial compounds mersalyl (10(-5) mol/l) and CH3ClHg (10(-3) mol/l), as well as La3+ (10(-4) mol/l), vanadate (10(-4) mol/l), and saponin (50 micrograms/mg prot), abolished Mg-ATP-promoted 45Ca2+ uptake. In the absence of Mg2+, ATP did not provoke 45Ca2+ uptake. Using the purified smooth membrane fraction (F1) from the Ficoll-sucrose density gradient (enrichment of Na+-K+-ATPase specific activity by ninefold and of NADH-cytochrome c reductase by threefold as compared with total tissue homogenate), Mg-ATP-dependent 45Ca2+ uptake correlated better with Na+-K+-ATPase (r = 0.97) than with the smooth endoplasmic marker NADH-cytochrome c reductase (r = 0.52). No correlation was found with RNA, the marker for rough endoplasmic reticulum. We conclude that pancreatic plasma membranes contain a Ca2+-Mg2+-ATPase that represents the Ca2+ extrusion system from acinar cells. It is also possible that vesicular membrane structures associated with the plasma membrane, or endocytotic plasma membrane vesicles, take up Ca2+ and represent an intracellular Ca2+ pool.
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PMID:Characterization of Mg-ATP-dependent Ca2+ transport in cat pancreatic microsomes. 613 52

A method is described for the isolation of endoplasmic reticulum and Golgi apparatus from hyperplastic liver nodules produced by discontinuous feeding of 2-acetylaminofluorene to male Wistar rats. The procedure involves three centrifugation steps and permits the separation of these cell components and their subfractions from the same sample of liver tissue as little as 1 g, wet weight. The fractions have been characterized by chemical, enzymatic, and morphological techniques and were found to be as pure as preparations from normal tissue. Furthermore, some of the characteristic histochemical features of hyperplastic liver nodules have been quantitated by biochemical methods in the fractions. Glucose-6-phosphatase activity in the endoplasmic reticulum subfractions of nodules is approximately 15% of the corresponding value in normal livers, whereas the activity of reduced nicotinamide adenine dinucleotide phosphate: cytochrome c reductase is reduced to 85% of the normal activity. The amount of cytochrome P-450 in nodular membranes as measured by differential spectroscopy is 25% of the control, indicating a decreased Phase I activity in drug metabolism. A 5-fold increase in cytosolic glutathione S-transferase activity without change in the corresponding microsomal activity was detected in hepatocyte nodules in rat liver. The activity of gamma-glutamyltransferase is increased more than 20-fold in all membrane fractions prepared from nodular tissue. The cytosolic activity, which is very low in the normal liver, is similarly increased more than 20-fold. The membrane-associated gamma-glutamyltransferase seems to be an integral membrane protein which cannot be washed away from the membranes. Chemically, membranes from nodules have phospholipid and cholesterol:protein ratios as found in membranes from normal liver tissue. However, the composition of individual phospholipids is changed with a 2-fold increase in nodular phosphatidylinositol and a slight decrease in phosphatidylcholine content in nodular membranes. The amount of endoplasmic reticulum membranes is of the same magnitude as in normal liver, although the smooth-surfaced component constitutes almost 60% of the isolated endoplasmic reticulum marker enzymes in nodules, compared with only 32% in preparations from normal tissue. The albumin contents of nodular and normal microsomal and Golgi membrane preparations are similar, indicating a normal synthesis of albumin by nodular tissue.
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PMID:Isolation and characterization of endoplasmic reticulum and Golgi apparatus from hepatocyte nodules in male wistar rats. 618 97

We studied the morphologic appearance of alcoholic hyalin (AH)-containing hepatocytes in liver biopsies from 14 patients with alcoholic liver disease. Most hepatocytes had a characteristic appearance. The cells were swollen and hydropic with an intact cell membrane. The mitochondria had variable-sized cristae which were both shortened and elongated. The smooth endoplasmic reticulum was markedly decreased. The rough endoplasmic reticulum was bizarre, with detachment of the ribosomes that surrounded the AH. The hepatocytes that contained AH bodies had lost almost all the glucose-6-phosphate activity but had variable amounts of succinic dehydrogenase and diphosphopyridine nucleotide diaphorase activities. The neutrophils admixed with mononuclear cells attached themselves to the hepatocytes and then invaginated into the hepatocytic cytoplasm with focal lysis of the cell membrane mediated via the release of neutrophilic lysosomes. The distortion of protein-synthesizing organelles and decrease in glucose-6-phosphatase activity suggest that the AH-containing hepatocyte is metabolically decompensated. The final cell death may be related to the neutrophilic attack, rather than the metabolic derangement.
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PMID:Alcoholic hyalin-containing hepatocytes--a characteristic morphologic appearance. 620 13


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