Isolation and partial characterization of human erythrocyte membrane NADH: (acceptor) oxidoreductase.
The NADH: (acceptor) oxidoreductase (EC 1.6.99.3) was isolated from human erythrocyte ghosts by a procedure including Triton X-100 solubilization, affinity chromatography on an NAD+-Sepharose 4B column, ammonium sulfate precipitation, and isoelectric focusing. This enzyme preparation was characterized by a single band on the urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by a single precipitin line with its corresponding antiserum on double diffusion and immunoelectrophoresis. A 103-fold purification indicates that the oxidoreductase represents approximately 1% of the ghost protein mass. The specific activity of the purified enzyme was 112 units/mg protein. The pH optimum was 6.8 and the isoelectric point, pI, was 6.6 The oxidoreductase has a specificity for NADH as a cofactor. The NADPH was ineffective as a reducing agent. The enzyme activity was strongly temperature-dependent, displaying maximal activity between 35 and 40 degrees C. The energy of activation was 4.9 kcal. The enzyme activity was inhibited by sulfhydryl reagents, anionic detergents, and divalent ions. The amino acid composition of the purified enzyme is characterized by the presence of all common amino acids including half-cystine and tryptophan. The results of carbohydrate and lipid analyses indicated that the oxidoreductase is a glycolipoprotein with fucose, galactose, mannose, and glucosamine as the sugar components and cholesterol and sphingomyelin as the lipid constituents. The apparent subunit molecular weight estimated by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol was 40,000. The antiserum completely inhibited the enzymic activity at the equivalence point. We suggest that the membrane-bound NADH: (acceptor) oxidoreductase might be a transmembrane protein.
D000937:Antigen-Antibody Reactions; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D006023:Glycoproteins / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000097:blood; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification
D002241:Carbohydrates; D006023:Glycoproteins; D008565:Membrane Proteins; D009247:NADH, NADPH Oxidoreductases