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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A flavoprotein catalyzing the reduction of cytochrome c by NADPH was solubilized and purified from microsomes of yeast grown anaerobically. The cytochrome c reductase had an apparent molecular weight of 70,000 daltons and contained one mole each of FAD and FMN per mole of enzyme. The reductase could reduce some redox dyes as well as cytochrome c, but could not catalyze the reduction of cytochrome b5. The reductase preparation also catalyzed the oxidation of NADPH with molecular oxygen in the presence of a catalytic amount of 2-methyl-1,4-naphthoquinone (menadione). The Michaelis constants of the reductase for NADPH and cytochrome c were determined to be 32.4 and 3.4 micron M, respectively, and the optimal pH for cytochrome c reduction was 7.8 to 8.0. It was concluded that yeast NADPH-cytochrome c reductase is in many respects similar to the liver microsomal reductase which acts as an NADPH-cytochrome P-450 reductase [EC 1.6.2.4].
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PMID:Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase. 1 31

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.
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PMID:Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes. 17 49

Vesicles were prepared from a 9:1 (mole/mol) mixture of dipalmitoyl phosphatidylcholine and the radioactively labeled phospholipids, 1-palmitoyl-2-omega-(m-diazirinophenoxy)undecanoyl-sn-glycero-3-phosphocholine (PC-I) or 1-palmitoyl-2-omega-(2-diazo-3,3,3-trifluropropionyloxy)lauroyl-sn- glycero-3-phosphocholine (PC-II). Rabbit liver cytochrome b5 was inserted into these vesicles spontaneously and the resulting vesicles containing the cytochrome b5 in the transferable form were photolyzed. Cytochrome b5 containing covalently cross-linked phospholipids was isolated by Sephadex LH-60 column chromatography using ethanol/formic acid as the solvent. Of the total radioactivity, 4.6% (PC-I) or 11.3% (PC-II) was linked to the protein; of the former, up to 51% was base-labile, while in the latter, 22% was base-labile. The sites of cross-linking of PC-I to the protein were investigated by fragmentation with trypsin, Staphylococcus aureas V8 protease, CNBr, and o-iodosobenzoic acid followed by Sephadex LH-60 chromatography and Edman sequencing (solid phase) of the appropriate fragments. The distribution of cross-linking was broad (Ser-104 to Met-130), showing a bell-shaped pattern with a significant peak at Ser-118. The labeling pattern is consistent with the previously proposed loop-back model for the membranous segment in the transferable form of cytochrome b5.
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PMID:The membrane-embedded segment of cytochrome b5 as studied by cross-linking with photoactivatable phospholipids. 634 39

The transbilayer distribution of phosphatidylethanolamine was assessed in phosphatidylcholine-phosphatidylethanolamine vesicles that contained various amounts of cytochrome b5. The small vesicles, made by sonication, and the large vesicles, made by ethanol injection, were fractionated by centrifugation before cytochrome b5 was asymmetrically incorporated into the bilayer. The mole ratio of phospholipid to protein ranged from 280 to 560 in the small vesicles and from 100 to 500 in the large vesicles. The phosphatidylethanolamine distribution, determined by chemical labeling with trinitrobenzenesulfonic acid, was assessed in vesicles the contained intact cytochrome b5 molecules and in vesicles where only the hydrophobic tail remained associated with the bilayer. At every phospholipid to protein ratio examined, the transbilayer distribution of phosphatidylethanolamine in either the small or large unilamellar vesicles was not significantly different from the distribution in control vesicles that contained no protein. Ethanol was added to some cytochrome b5-vesicle preparations (20% v/v) in an attempt to facilitate rearrangement of the phospholipids. No differences in the transbilayer distribution were observed. These results are discussed in terms of transbilayer equilibrium and the perturbation induced by the protein.
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PMID:Effect of cytochrome b5 on the transbilayer distribution of phospholipids in model membranes. 710 93

The problem of determining small but significant amounts of carbohydrates, in purified proteins, has been studied using the membrane protein, cytochrome b5. A newly developed method that involves direct gas chromatography-mass spectrometry of sugars obtained by hydrolysis of proteins purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) allows the identification and determination of small amounts of carbohydrates (e.g., 20 micrograms of glycoprotein containing a minimum of 0.1% monosaccharide), even in the presence of relatively high amounts of impurities. Application of this method to cytochrome b5 fragments obtained by tryptic digestion from rat liver microsomes and purified by combined gel filtration and ion exchange chromatography, followed by SDS PAGE, has consistently yielded values below 0.07 mol of the individual sugars and aminosugars per mole cytochrome b5. It is concluded that cytochrome b5, at least its trypsin-released major amino-terminal fragment, is not constitutively glycosylated.
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PMID:Absence of sugars in electrophoretically purified cytochrome b5 demonstrated by combined gas chromatography-mass spectrometry. 725 67

In recent years many studies have been directed toward the elucidation of the interaction mechanisms within protein-protein complexes. One of the best studies protein-protein complexes has been the cytochrome b5 and cytochrome c electron transfer pair. Thermodynamic information about the association process has been obtained through methods which indirectly measure the binding between the proteins. We report here the use of Isothermal Titration Calorimetry to characterize the association of Rat cytochrome b5 and Horse cytochrome c. The association is accompanied by an unfavorable enthalpy change (+1.0 +/-0.1 Kcal/mole) and a large stabilizing change in entropy (33.9 +/-0.6 eu).
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PMID:Thermodynamic characterization of the interaction between cytochrome b5 and cytochrome c. 757 8

To determine the effect of cholesterol and lipid packing on the solubility of membrane proteins in bilayers, cytochrome b5 incorporation into phosphatidylcholine (PC) liposomes was determined as a function of bilayer curvature (SUVs versus LUVs), fatty acyl chain composition, and cholesterol content. The equilibrium affinity constant for the formation of a 1:1 b5/PC complex, Kp, and the number of PC's per "site" at saturation, n, were determined from binding isotherms, which were obtained by measuring the increase in intrinsic tryptophan fluorescence. With LUVs, n was also determined directly by gel filtration. The following results were obtained: (1) Both Kp and the saturating level of b5 binding, s (n-1), are significantly greater for SUVs than for LUVs. In LUVs, a binding site must consist of several surrounding lipid layers. (2) Cholesterol reduces Kp and s by factors that range from 1 to > 100. Binding inhibition is highly sensitive to the liposome size and to the fatty acyl composition of the PC; the latter correlates with the condensing effects on PC: C1satC2mono > C1satC2di approximately natural mixtures > C1unsat-C2unsat. (3) With POPC LUVs, the binding inhibition was 3.6-, 1.4- and 17-fold within the ranges of 0-20, 20-33, and 33-50 mole percent cholesterol, respectively. (4) The equilibrium binding constant to SUVs is greater for liposomes that are prepared from natural PC mixtures than for vesicles of a single synthetic phospholipid. The reductions in b5 binding correlate with reductions in bilayer free volume, which were calculated from monolayer studies of the lipid mixtures. The sensitivity of liposome saturability to bilayer curvature, fatty acyl chain composition, and cholesterol content may account for the disparate results among previous studies of cholesterol-protein interactions. A more significant implication is that in biological membranes with high levels of cholesterol, subtle variations in the fatty acyl chain composition could substantially affect the solubility and physical states of integral membrane proteins.
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PMID:Effect of cholesterol, fatty acyl chain composition, and bilayer curvature on the interaction of cytochrome b5 with liposomes of phosphatidylcholines. 789 81

The fluorescence of a membrane-bound tryptophan derivative (tryptophan octyl ester, TOE) has been examined as a model for tryptophan fluorescence from proteins in membrane environments. The depth-dependent fluorescence quenching of TOE by brominated lipids was found to proceed via a dynamic mechanism with vertical fluctuations playing a central role in the process. The activation energy for the quenching was estimated to be 1.3 kcal/mole. The data were analyzed using the distribution analysis (DA) method, which extends the conventional parallax method to account more realistically for the transbilayer distributions of both probe and quencher and for possible variations in the probe's accessibility. DA provides a better fit than the parallax method to data collected with TOE in membranes formed of lipids brominated at either the 4,5, the 6,7, the 9,10, or the 11,12 positions of the sn-2 acyl chain. DA yields information on the fluorophore's most probable depth in the membrane, its conformational heterogeneity, and its accessibility to the lipid phase. Previously reported data on cytochrome b5 and melittin were reanalyzed together with data obtained with TOE. This new analysis demonstrates conformational heterogeneity in melittin and provides estimates of the freedom of motion and exposure to the lipid phase of membrane-embedded tryptophans of cytochrome b5.
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PMID:Fluorescence of membrane-bound tryptophan octyl ester: a model for studying intrinsic fluorescence of protein-membrane interactions. 1152 96