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

---

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

A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20-50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30-33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 micromole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with (125)I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells.
...
PMID:Isolation of plasma membrane fragments from HeLa cells. 423 70

Zonal centrifugation has been used to isolate a fraction from bovine liver which appears to be derived from the Golgi apparatus. Morphologically, the fraction consists mainly of sacs and tubular elements. Spherical inclusions, probably lipoproteins, are occasionally seen in negative stains of this material. The preparation is biochemically unique. UDP-galactose:N-acetyl glucosamine, galactosyl transferase activity is concentrated about 40-fold in this fraction compared to the homogenate. Rotenone- or antimycin-insensitive DPNH- or TPNH- cytochrome c reductase activities are 60-80% of the level of activities found in microsomes. Purified organelles from bovine liver such as plasma membranes, rough microsomes, mitochondria and nuclei have negligible levels of galactosyl transferase. Some activity is present in smooth microsomes but at a level compatible with the possible presence of Golgi membranes in this fraction. The Golgi fraction does not contain appreciable amounts of enzymes such as ATPase, 5'-nucleotidase, glycosidase, glucose-6-phosphatase, acid phosphatase, or succinate-cytochrome c reductase. Similar fractions isolated from bovine epididymis also have very high levels of galactosyl transferase. The fraction is heavily osmicated when incubated for long periods of time at elevated temperatures, a characteristic property of Golgi membranes.
...
PMID:Isolation and characterization of Golgi membranes from bovine liver. 424 7

A rapid method of preparing plasma membranes from isolated fat cells is described. After homogenization of the cells, various fractions were isolated by differential centrifugation and linear gradients. Ficoll gradients were preferred because total preparation time was under 3 hr. The density of the plasma membranes was 1.14 in sucrose. The plasma membrane fraction was virtually uncontaminated by nuclei but contained 10% of the mitochondrial succinic dehydrogenase activity and 25-30% of the RNA and reduced nicotinamide adenine dinucleotide cytochrome c reductase activity of the microsomal fraction. Part of the RNA and NADH-cytochrome c reductase activity was believed to be native to the plasma membrane or to the attached endoplasmic reticulum membranes demonstrated by electron microscopy. The adenyl cyclase activity of the plasma membrane fraction was five times that of Rodbell's "ghost" preparation and retained sensitivity to epinephrine. The plasma membrane ATPase activity was five times that of the homogenate and microsomal fractions. Electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.
...
PMID:Preparation and characterization of a plasma membrane fraction from isolated fat cells. 424 33

1. One week after denervation several biochemical characteristics of the fast extensor digitorum longus and slow soleus muscles from adult rats were investigated and compared with the characteristics of the corresponding unoperated contralateral muscles. 2. After these short periods of denervation-induced atrophy, the isolated myosins showed unchanged ATPase (adenosine triphosphatase) activities, but there was the expected difference between fast and slow muscle. 3. The specific activities of several soluble enzymes and their characteristic patterns were found to be only slightly modified in both the extensor and soleus muscles after denervation, as were most of the activities measured in the isolated mitochondria. 4. The most significant modifications were in the isolated sarcoplasmic reticulum, and appeared to be specific to either slow or fast muscle. 5. Denervation of slow muscle led to a marked increase of Ca(2+)-transport rates, and of the specific activity of the Mg(2+)-activated K(+)-modulated Ca(2+)-stimulated ATPase, together with changes in the polyacrylamide-electrophoretic profiles of the microsomal membrane protein. Transformation of these several properties of slow muscle sarcoplasmic reticulum to those of fast muscle sarcoplasmic reticulum was further substantiated by electron-microscopic analysis after negative staining. Control experiments with tenotomized soleus muscle gave negative results. 6. The isolated sarcoplasmic reticulum from fast muscle showed a slight diminution of ATPase-linked Ca(2+)-transport activity and a selective increase of rotenone-insensitive NADH-cytochrome c reductase activity, in addition to a greater emphasis on slow-type electrophoretic components of the structural membrane protein. 7. The significance of these results in relation to specific differentiating influences from motor nerves is discussed.
...
PMID:Early biochemical consequences of denervation in fast and slow skeletal muscles and their relationship to neural control over muscle differentiation. 426 59

Plasma membranes from KB cells were isolated by the method of latex bead ingestion and were compared with those obtained by the ZnCl(2) method. Optimal conditions for bead uptake and the isolation procedure employing discontinuous sucrose gradient centrifugation are described. All steps of preparative procedure were monitored by electron microscopy and specific enzyme activities. The plasma membrane fraction obtained by both methods is characterized by the presence of the Na(+) + K(+)-activated ATPase and 5'-nucleotidase, and contains NADPH-cytochrome c reductase and cytochrome b(5). The latter two enzymes are also present in lower concentrations in the microsomal fraction. Unlike microsomes which are devoid of the Na(+) + K(+)-activated ATPase and which contain only traces of 5'-nucleotidase activity, the plasma membrane fraction contains only trace amounts of the rotenone-insensitive NADH-cytochrome c reductase but no cytochrome P-450, both of which are mainly microsomal components. Morphologically the plasma membrane fraction isolated by the latex bead method is composed of vesicles of 0.1-0.3 microm in diameter. On the basis of the biochemical and morphological criteria presented, it is concluded that the plasma membrane fraction isolated by the above methods are of high degree of purity.
...
PMID:Isolation and properties of the plasma membrane of KB cells. 428 30

Partially purified plasma membranes were obtained from chick-embryo muscle cells grown in tissue culture. The purification procedure involved homogenization in buffered isotonic sucrose followed by differential and sucrose density gradient centrifugations. The activities of five plasma-membrane markers, as well as microsomal and mitochondrial markers, were followed throughout the purification. When cultures were labeled with [(125)I]alpha-bungarotoxin, which binds to the surface of cultured muscle cells, the distributions of bound alpha-bungarotoxin and Na(+),K(+)-ATPase (EC 3.6.1.3) activity were nearly identical. The activities of these two plasma-membrane markers were maximal in the upper two fractions of the sucrose density gradient and were purified 5- to 7-fold with respect to total particulate protein. These fractions contained 20-30% of the Na(+),K(+)-ATPase activity and bound alpha-bungarotoxin, 4% of the microsomal marker TPNH-dependent cytochrome c reductase, 0.2% of the mitochondrial marker succinate-dependent cytochrome c reductase, 2.7% of the cellular RNA, and 0.02% of the DNA. The activity of the commonly used plasma-membrane marker, 5'-nucleotidase (EC 3.1.3.5), was low in the upper two sucrose gradient fractions and was maximal in a more dense fraction. The distributions of the other two plasma-membrane markers, leucyl beta-naphthylamidase and phosphodiesterase I, were intermediate between Na(+),K(+)-ATPase and 5'-nucleotidase. The distributions of all markers were similar in preparations from cultures containing mononucleated myogenic cells, multinucleated myotubes, fibroblasts, or all three cell types. Modification of the procedure to include homogenization in the absence of sucrose resulted in a 3.4-fold purification of the membranes containing 5'-nucleotidase, which were shifted to a lower density.
...
PMID:Plasma membranes from cultured muscle cells: isolation procedure and separation of putative plasma-membrane marker enzymes. 436 82

Sarcolemmal and microsomal membranes prepared from adult canine cardiac myocytes (sarcolemmal Na+, K+-ATPase = 71.8 mumol/mg per hr and microsomal rotenone-insensitive NADH cytochrome c reductase = 114 mumol/mg per hr) were each preincubated at 37 degrees C in the presence of a free radical generating system consisting of dihydroxyfumarate and Fe -ADP; loss of the Na+, K+-ATPase and reductase activities, as well as the associated increases in lipid peroxidation, measured by malondialdehyde formation, were temporally correlated in both systems. The ATPase was inhibited 70% when the malondialdehyde was 71 nmol/mg protein at 20 minutes and 90% when malondialdehyde was 138 nmol/mg protein at 90 minutes. Inhibition of reductase activity occurred more gradually, displaying a 27% loss of activity when malondialdehyde reached 34 nmol/mg protein at 20 minutes and 60% with a malondialdehyde value of 67 nmol/mg protein at 90 minutes. The greater susceptibility of the sarcolemma to free radical-induced membrane damage may be due to the higher content of unsaturated fatty acids in this membrane, compared to microsomes.
...
PMID:Differential sensitivity of canine cardiac sarcolemmal and microsomal enzymes to inhibition by free radical-induced lipid peroxidation. 608 70

The maximum Gibbs free energies of reverse electron transfer from succinate to NAD+ and from cytochrome c to fumarate driven by ATP hydrolysis in submitochondrial particles from beef heart were measured as a function of the Gibbs free energy of ATP hydrolysis. The ratio of the energies delta G'redox/delta G'ATP was 1.40 from succinate to NAD+ and 0.89 from cytochrome c to succinate. The ratio, equivalent to a thermodynamic P/2e-ratio, was dependent on whether the electrochemical proton gradient was primarily a membrane potential or a pH gradient for the cytochrome c to fumarate reaction. The results are consistent with H+/ATP = 3 for F1 ATPase, H+/2e- = 4 for NADH-CoQ reductase, and H+(matrix)/2e- = 2 for succinate-cytochrome c reductase.
...
PMID:Energetics of ATP-driven reverse electron transfer from cytochrome c to fumarate and from succinate to NAD in submitochondrial particles. 608 93

We have studied a 17-year-old girl with lactic acidosis (3-18 mEq/liter) and progressive muscle weakness since 9 years of age. Morphological findings in muscle were of a typical ragged red myopathy with multiple collections of bizarre mitochondria, some containing paracrystalline inclusions. The carnitine content of serum and muscle was normal, as were the activities of carnitine palmitoyltransferase, carnitine octanoyltransferase, and carnitine acetyltransferase in the patient's muscle. Measurement of the enzymes of oxidative phosphorylation in both crude muscle homogenates and mitochondrial fractions showed close to normal activities of cytochrome c oxidase, succinate dehydrogenase, and ATPase. In contrast, succinate cytochrome c reductase activity was greatly reduced in the patient, being 0.035 mumol/min/g tissue in whole muscle (controls 1.16 +/- 0.47 mumol/min/g tissue) and 8 nmol/min/mg protein in the mitochondria (control, 340 nmol/min/mg protein). Rotenonesensitive NADH-cytochrome c reductase was also undetectable in the patient's mitochondria. Spectral analysis of cytochromes showed decrease of reducible cytochrome b to 16% of the control. These results indicate a defect of ubiquinol-cytochrome c reductase or the cytochrome bc1 segment (complex III) of the electron transport chain. Antibody-binding studies of the individual components of complex III showed additional deficiencies of core proteins I and II and peptide VI, indicating a more widespread defect of complex III than was evident from spectral analysis and enzyme activity measurements alone. Urine organic acid analysis after fasting and following a medium chain triglyceride load showed unusually high levels of lactate and 3-hydroxybutyrate, lower than expected levels of acetoacetate and dicarboxylic acids, and the presence of several other metabolites suggesting a disturbed citric acid cycle and redox state.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Lactic acidosis and mitochondrial myopathy associated with deficiency of several components of complex III of the respiratory chain. 609 35

Rat astrocytes in primary cultures were employed to isolate the plasma membrane. The method for the isolation of plasma membrane was based on the capacity of the cytoskeleton to adhere to the substratum entrapping intracellular organelles during freezing-thawing cycle performed on the cell. By washing the 'surface adherent framework', the untrapped plasma membrane were recovered and density equilibrium centrifugation resulted in the isolated membrane. The isolated plasma membrane was characterized on the basis of a variety of marker enzymes positive to the plasma membrane such as (Na+ + K+)-ATPase or 5'-nucleotidase as well as the lack of conventional markers of other endomembranes. Ultrastructurally the membranes, as isolated here, were mainly vesicular in nature. The isolated plasma membrane was devoid of the dehydrogenase responsible for NADH-cytochrome c reductase activity. However, NADH-ferricyanide reductase activity and the dehydrogenase system catalyzing the transfer of reducing equivalents from NADH or NADPH to dichloroindophenol seems plasma membrane redox system. The identical specific activity employing dichloroindophenol as an electron acceptor with NADH or NADPH as donor indicate a DT-diaphorase (EC 1.6.99.2) like activity in the astrocytes plasma membrane.
...
PMID:Plasma membrane isolated from astrocytes in primary cultures. Its acceptor oxidoreductase properties. 609 77


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---