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 non-ionic detergent octyl glucoside solubilizes a substantial amount of Streptococcus faecalis membrane protein without loss of the monitored enzyme activities. A secondary detergent, dioctanoyl phophatidycholine, appears to increase the yield of solubilized material. In addition, the effect of ionic strength indicates that it may be possible to selectively extract groups of membrane proteins by their characteristic solubility at different ionic strengths. The solubilized membrane-associated enzymes, ATPase and NADH dehydrogenase, enter polyacrylamide gels as distict species. Electrophoretic studies suggest that there are two membrane-associated ATPase in the Streptococcus faecalis, one which dissociates from the membrane in the absence of Mg-2+ ions and the other which remains particulate until solubilized by detergents. Octyl glucoside can be easily removed from a solution containing solubilized proteins and lipid by dialysis.
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PMID:Solubilization of bacterial membrane proteins using alkyl glucosides and dioctanoyl phosphatidylcholine. 12 71

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.
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PMID:Functional mosaicism of membrane proteins in vesicles of Escherichia coli. 19 Feb 12

This paper reviews mechanisms by which the rate of synthesis of subunits of mitochondrial inner membrane protein complexes and the assembly of these subunits are co-ordinated. Current models are evaluated and critically discussed in the light of some recent evidences. The focus is on the incorporation of cytoplasmically-synthesized cytochrome c oxidase subunits in the development of a newer model, which introduces some twists into a combination of several current ideas. A mechanism which governs both organized assembly and the co-ordination of rates of polypeptide synthesis is illustrated and the principles of the model are applied to the elucidation of some odd features of certain mutants. The possibilities that mitochondrial ATPase and cytochrome c reductase may also be synthesized and assembled according to this model are discussed.
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PMID:Biosynthesis of mitochondrial membrane proteins: co-ordination with special reference to cytochrome c oxidase. 20 73

Microsomal NADH-cytochrome b5 reductase is an amphiphilic protein consisting of a hydrophilic (catalytic) region and a hydrophobic (membrane-binding) segment. Digestion of the reductase purified from rabbit liver microsomes with carboxypeptidase Y (CPY), but not with aminopeptidases, resulted in the abolishment of the capacities of the reductase to bind to phosphatidylcholine liposomes and to reconstitute an active NADH-cytochrome c reductase system upon mixing with cytochrome b5. The NADH-ferricyanide reductase activity of the flavoprotein was, however, inactivated only slightly by the CPY digestion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analyses indicated that the CPY treatment removed about 30 amino acid residues from the tcooh terminus of the reductase and that about 70% of the amino acids released were hydrophobic. It is concluded that the hydrophobic region of the reductase, responsible for both membrane binding and effective reconstitution of NADH-cytochrome c reductase activity, is located at the COOH-terminal portion of the molecule. No NH2-terminal residue could be detected in the intact and CPY-modified reductase preparations. The location of the hydrophobic, membrane-binding segment at the COOH-terminal end and the masked NH2 terminus have also been reported for cytochrome b5, another microsomal membrane protein.
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PMID:Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase: location of the hydrophobic, membrane-binding region at the carboxyl-terminal end and the masked amino terminus. 21 Jul 82

Cells of Rhodopseudomonas capsulata, strain 37b4, leu-, precultivated anaerobically under low light intensity, were exposed to high light intensity (2000 W.m-2). The cells grew with a mass doubling time of 3 h. The synthesis of bacteriochlorophyll (BChl) began after two doublings of cell mass. Reaction center and light-harvesting BChl I (B-875) were the main constituents of the photosynthetic apparatus incorporated into the membrane. The size of the photosynthetic unit (total BChl/reaction center) decreased and light-harvesting BChl I became the dominating BChl species. Concomitant with the appearance of the different spectral forms of BChl the respective proteins were incorporated into the membrane, i.e. the three reaction center polypeptides, the polypeptide associated with light-harvesting BChl I, the two polypeptides associated with BChl II. A polypeptide of an apparent molecular weight of 45 000 was also incorporated. A lowering of the light intensity to 7 W.m-2 resulted in a lag phase of growth for 6 h. Afterwards, the time for doubling of cell mass was 11 h. The concentration of all three BChl complexes (reaction center, light-harvesting BChl I and II complexes)/cell and per membrane protein increased immediately. Also the size of the photosynthetic unit and the amount of intracytoplasmic membranes/cell increased. The activities of photophosphorylation, succinate dehydrogenase, NADH dehydrogenase and NADH oxidation (respiratory chain)/membrane protein are higher in membrane preparations isolated from cells grown at high light intensities than in such preparations from cells grown at low light intensities.
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PMID:Effects of light intensity on membrane differentiation in Rhodopseudomonas capsulata. 48 32

Sheep erythrocyte membranes have been shown in this laboratory to undergo spontaneous vesiculation when incubated at 4 degrees, fractionating into two bands in dextran gradients (R. McGuire and R. Barber, submitted for publication). While vesicles were observed to be formed in several solvent systems, incubation in the presence of complexors to remove divalent cations was found to be the most efficient method for both vesicle formation and their detachment from the residual membrane. We report here on the characterization of these vesicles formed by spontaneous vesiculation. In the presence of a hypotnoic buffer containing 1 mM EDTA, vesicle production proceeds linearly up to 50 hours and declines, reaching its maximum at 72 hours with up to 20% of the total membrane protein found in the upper band. This upper band is shown in electron micrographs to be composed chiefly of closed vesicles, while the particles in the lower band appear morphologically similar to the original ghosts. Total phospholipid phosphorus and cholesterol in the vesicles are enriched to the same extent, giving a lipid to protein ratio of 2 times that found for whole ghosts. The vesicles contain the same individual phospholipids as the ghosts. The protein composition of these vesicles is unique, in that they are almost depleted in the known extrinsic membrane proteins, while containing practically all types of the various glycoproteins of the original membrane. The two main intrinsic membrane proteins (with apparent molecular weights of 160,000 and 100,000) are found almost exclusively in the vesicles, virtually depleted in the residual ghost-like particles. The protein with 160,000 molecular weight is shown here to be a glycoprotein, giving an anomalous molecular weight on sodium dodecyl sulfate gels and having a molecular weight of approximately 50,000 after lipid extraction. This same glycoprotein appears to fractionate with acetylcholinesterase. From the accessibilities of the substrates to the membrane acetylcholinesterase and NADH-diaphorase, it is concluded that the vesicles are right-side-out and sealed to small molecules. There are more membrane sialic acid residues accessible to neuraminidase in the vesicles (in terms of number of residues/mg og membrane protein) than in ghosts, further supporting the conclustion that these vesicles have a normal orientation and are enriched in glycoproteins. The specific activity of acetylcholinesterase in the vesicles is increased 5- to 6-fold over that found in the original ghosts and almost 20-fold over that in the residual ghost-like particles. Consequently, spontaneous vesiculation occurs simultaneously with the enrichement of specific membrane proteins in certain regions of the lipid bilayer. It is postulated that these domains in the membrane, containing clusters of specific intrinsic membrane proteins, bud out and subsequently release glycoprotein-enriched lipid vesicles.
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PMID:Glycoprotein-enriched vesicles from sheep erythrocyte ghosts obtained by spontaneous vesiculation. 93 96

1. Anti-heart mitochondria autoantibodies were developed in serum from dogs following experimental myocardial infarction. 2. Heart mitochondria frozen and thawed repeatedly in a sucrose/Tris-chloride buffer retained both their functional integrity as measured by the respiratory control ratio and their ability to serve as an antigen in a complement fixation test. Mitochondria frozen and thawed in a potassium chloride/Tris-chloride buffer lost both their functional integrity and their autoantigenic activity after one freeze-thaw cycle. 3. Extraction of the heart mitochondria with acetone/water mixtures to remove phospholipids from the membrane led to a complete loss of the ability of the mitochondria to react in the complement fixation test but did not affect the ability of the membranes to bind autoantibody in absorption experiments. 4. Treatment of the mitochondrial membranes with increasing concentrations of trypsin caused a loss of up to approximately 50% of the membrane protein with a gradual decrease in the autoantigenic activity of the membrane without impairment of the ability of the membrane to bind autoantibody. 5. Removal of up to 90% of the sialic acid of the mitochondrial membrane with neuraminidase resulted in a considerable increase in the complement-fixing autoantigenic activity of the membrane without changing the apparent ability of the membrane to bind autoantibody in absorption experiments. 6. Exposure of mitochondrial membranes to autoantibody and complement caused an inhibition of both an inner mitochondrial membrane enzyme, i.e. cytochrome oxidase (48%) and an outer mitochondrial membrane enzyme, i.e. NADH cytochrome c reductase (rotenone insensitive) (37%).
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PMID:Characterization of autoantigenic sites on isolated dog heart mitochondria. 118 45

Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I-sensitive component(s). In the presence of a concentration of Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2- production by Ph2I. The O2(-)-generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2- production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.
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PMID:Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation. 132 36

The amino-terminal region of a 70 kDa mitochondrial outer membrane protein of yeast and the presequence of cytochrome c1, an inner membrane protein exposed to the intermembrane space, are thought to be responsible for localizing the proteins in their final destinations after synthesis in the cytosol. Gene fusion experiments were used to identify signals that are responsible for protein sorting between the outer and inner mitochondrial membranes. The submitochondrial localization of cytochrome c1 whose presequence was replaced by the amino-terminal region of the 70 kDa mitochondrial outer membrane protein has been investigated. We have also used an in vivo complementation assay to determine whether or not a 70k-cyt c1 fusion protein is functional. Both the first half and all of the presequence of cytochrome c1 can be replaced by the amino-terminal 12 or 29 residues of the 70 kDa protein for transport to the inner membrane and functional assembly into succinate-cytochrome c reductase. However, replacements by the amino-terminal 61 residues of the 70 kDa protein result in exclusive localization of the fusion proteins to the outer membrane, and the fusions cannot be assembled into the enzyme complex. These data indicate that a mitochondrial targeting signal alone is sufficient to direct cytochrome c1 of mature size to the inner membrane.
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PMID:Protein sorting between the outer and inner mitochondrial membranes: submitochondrial localization of cytochrome c1 whose presequence is replaced by the amino-terminal region of a 70 kDa outer membrane protein. 255 Apr 19

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.
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PMID:Early biochemical consequences of denervation in fast and slow skeletal muscles and their relationship to neural control over muscle differentiation. 426 59


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