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H G Enoch

Publications and source records attributed to H G Enoch.

9 recordsLinked to original sources
PubMed1979

Formation and properties of 1000-A-diameter, single-bilayer phospholipid vesicles.

Two methods are reported for the formation of large, uniform-sized phospholipid vesicles. The methods involve the treatment of phospholipid, in the form of either small, sonicated vesicles or a dry lipid film, at a molar ratio of deoxycholate to phospholipid of 1:2. Subsequent removal of deoxycholate yields a stable preparation of vesicles. These vesicles are bounded by a single bilayer, have an average diameter of 1000 A, and are readily separated from sonicated vesicles (230 A) by gel filtration on Sepharose 4B. Since the 1000-A vesicles are capable of trapping enzymes and other macromolecules, they may prove valuable for the delivery of liposome-entrapped solutes to cells and for the localization of peptide segments of a spectrum of membrane-bound proteins.

Bile Acids and Salts↗
PubMed1977

Cytochrome b5 and cytochrome b5 reductase-phospholipid vesicles. Intervesicle protein transfer and oreintation factors in protein-protein interactions.

NADH-cytochrome b5 reductase readily binds to preformed phospholipid vesicles either below or above the phase transition temperature of the lipid and in the absence of detergents. The isolated vesicles are free of unbound reductase, and the lipid is present as small, closed bilayers (250 to 400 A in diameter) as indicated by gel filtration, density gradient centrifugation, and internal volume measurements with [3H]glucose. The order of substrate specificity of the bound reductase is: ferricyanide = cytochrome b5 bound to reductase vesicles (100%) greater than cytochrome b5 heme peptide (13%) greater than unbound cytochrome b5 (4.5%) greater than cytochrome b5 vesicles (0.1%). This indicates that a specific orientation of cytochrome b5 and reductase in the bilayer is required for optimal interaction. Protein transfer occurs between reductase vesicles and cytochrome b5 vesicles. The transfer is time-dependent (40 to 70% complete in 2 h), does not involve vesicle fusion, is most rapid at the phase transition temperature of the phospholipid, and appears to require a fluid bilayer.

Animals↗
PubMed1976

Mechanism of rat liver microsomal stearyl-CoA desaturase. Studies of the substrate specificity, enzyme-substrate interactions, and the function of lipid.

The three purified proteins which are required for microsomal stearyl-CoA desaturation, NADH-cytochrome b5 reductase, cytochrome b5, and desaturase, have been combined with egg lecithin or dimyristyl lecithin vesicles to reconstruct a functional electron transport system capable of utilizing NADH and O2 in the desaturation of stearyl-CoA. Such preparations appear to consist of phospholipid vesicles which contain the three proteins bound to the outer surface of the vesicles. Acyl-CoA derivatives containing 12 to 19 carbon fatty acyl chains are required for desaturase activity while derivatives containing 9 to 20 carbons are capable of binding to the enzyme. Shorter chain acyl-CoA derivatives, free CoA, and free fatty acids do not appear to bind to the enzyme. Inhibition and analog studies suggest that the methylene chain of stearyl-CoA assumes an eclipsed ("gauche") conformation at carbon atoms 9,10 in the enzyme-substrate complex. Furthermore, isotope rate effects obtained with deuterated stearyl-CoA derivatives indicate that hydrogen removal is the rate-limiting step of desaturation. Stearyl-CoA binds to pure liposomes and desaturase-containing liposomes, and it is this form of stearyl-CoA which appears to be the substrate for desaturase. The Arrhenius plots of desaturase activity obtained using desaturase bound to egg lecithin liposomes, in which the liquid crystalline to crystalline phase transition temperature is -5 degrees, was linear between 15 and 35 degrees, while that obtained using desaturase bound to dimyristyl lecithin liposomes showed a break at 24 degrees coinciding with the liquid crystalline to crystalline phase transition temperature for this lipid. The decrease observed in the deuterium isotope rate effect below the transition temperature indicates that a step in the reaction sequence other than hydrogen abstraction becomes rate-limiting when the lipid is in the crystalline state. In this system translational diffusion does not emerge as the rate-limiting step. The liposomes contained sufficient reductase and cytochrome b5 so that translational diffusion was not rate-limiting.

Animals↗
PubMed1975

The purification and properties of formate dehydrogenase and nitrate reductase from Escherichia coli.

The membrane-bound formate dehydrogenase of Escherichia coli grown anaerobically in the presence of nitrate was solubilized with deoxycholate and purified to near homogeneity. The purification procedure included ammonium sulfate fractionation and chromatography on Bio-Gel A-1.5m and DEAE Bio-Gel A in the presence of the nonionic detergent, Triton X-100. This detergent caused a significant decrease in the molecular weight of the soluble formate dehydrogenase complex and allowed the enzyme then to be resolved from other membrane components. Anaerobic conditions were required throughout due to the sensitivity of the enzyme to oxygen inactivation. Formate dehydrogenase was judged to be at least 93 to 99% pure by the following procedures: polyacrylamide gel electrophoresis in the presence of Triton X-100 and sodium dodecyl sulfate, gel filtration, and sedimentation velocity studies. The purified enzyme exists as a detergent-protein complex (0.20 +/- 0.03 g of Triton X-100/g of protein) which has an S20,w of 18.1 S and a Stokes radius of 76 A. This corresponds to a molecular weight of 590,000 +/- 59,000. The enzyme had an absorbance spectrum of a b-type cytochrome which could be completely reduced by formate. The heme content corresponds to an equivalent weight of 154,000 which suggests a tetrameric structure for the enzyme. Formate dehydrogenase was found to contain (in relative molar amounts): 1.0 heme, 0.95 molybdenum, 0.96 selenium, 14 non-heme iron, and 13 acid-labile sulfide. Neither FAD nor FMN could be detected. The enzyme contains three polypeptides, designated alpha, beta, and gamma, whose molecular weights were estimated by gel electrophoresis in the presence of sodium dodecyl sulfate to be 110,000, 32,000, and 20,000, respectively. After separation of the polypeptides by gel filtration in the presence of sodium dodecyl sulfate alpha, beta, and gamma were found in 1:1.2:0.55 molar ratios. A study of the enzyme obtained from cells grown with [75Se]selenite showed that only the alpha polypeptide contained significant amounts of selenium. The enzyme will catalyze the formate-dependent reduction of phenazine methosulfate, dichlorophenolindophenol, methylene blue, nitroblue tetrazolium, benzyl viologen, methyl viologen, ferricyanide, and coenzyme Q6. Cyanide, azide, p-hydroxymercuribenzoate, iodoacetamide, and oxygen inhibit the enzyme. The procedure which was designed for the purification of formate dehydrogenase also yields a highly purified preparation of nitrate reductase. This nitrate reductase has been shown to contain significant amounts of heme (Enoch, H. G., and Lester, R. L. (1974) Biochem. Biophys. Res Commun. 61,1234-1241). The enzyme contains three polypeptides with molecular weights of 155,000, 63,000, and 19,000. When measured in the presence of Trition X-100 the Stokes radius of nitrate reductase is 75 A and the S20,w is 16 S which corresponds to a molecular weight of 498,000.

Aldehyde Oxidoreductases↗