NADH dehydrogenase from bovine neutrophil membranes. Purification and properties.
A membrane-associated NADH dehydrogenase from beef neutrophils was purified to homogeneity, using detergent (cholate plus Triton X-100) extraction and chromatography on DEAE-Sepharose CL-6B, agarose-hexane-NAD, and hydroxylapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 17,500, but the enzyme was highly aggregated (Mr greater than 450,000) in nondenaturing gels containing 0.1% Triton X-100. The protein band in nondenaturing gels was also stained for activity using NADH and nitro blue tetrazolium. The enzyme showed greatest electron acceptor activity with ferricyanide (100%), followed by cytochrome c (3.5%), dichloroindophenol (2.7%), and cytochrome b5 (0.34%). No activity was seen with oxygen. The Km values for NADH and ferricyanide were 18 and 9.5 microM, respectively, and NAD+ was a weak competitive inhibitor (Ki = 118 microM). No activity was seen with NADPH. No effects were seen with mitochondrial respiratory inhibitors such as azide, cyanide, or rotenone, but p-chloromercuribenzoate was strongly inhibitory and N-ethylmaleimide was weakly inhibitory. No free flavin was detectable in enzyme preparations. Based upon kinetic, physical, and inhibition properties, this NADH dehydrogenase differs from those previously described in microsomes and erythrocyte plasma membrane.
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003579:Cytochrome Reductases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005292:Ferricyanides / Q000378:metabolism; D005415:Flavins / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008566:Membranes / Q000201:enzymology; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009245:NADH Dehydrogenase / Q000302:isolation & purification*; D009504:Neutrophils / Q000201:enzymology*
D005292:Ferricyanides; D005415:Flavins; D009243:NAD; C007931:hexacyanoferrate III; D003579:Cytochrome Reductases; D009245:NADH Dehydrogenase