Abstract
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.
Highlights
A membrane-associatedNADH dehydrogenase from These workers found that cytochrome-c reductase activity beef neutrophils was purified to homogeneity, using was significantly stimulated by the addition of cytochrome b6 detergentextraction and to the membranes
The NADH dehydrogenase from beef neutrophil appearsto be distinct from previously reported enzymes from other sources with similar activities
It is differentiated from microsomal NADH-cytochrome-bS reductase by its molecular weight (17,500versus 43,000), apparent absence of flavin, and its kinetic and inhibition properties [19,20,21]
Summary
A membrane-associatedNADH dehydrogenase from These workers found that cytochrome-c reductase activity beef neutrophils was purified to homogeneity, using was significantly stimulated by the addition of cytochrome b6 detergent (cholate plus Triton X-100)extraction and to the membranes. Based on this finding plus the similar chromatographyon DEAE-SepharoseCL-GB, agarose- catalytic and inhibition properties of the neutrophil and mihexane-NAD,andhydroxylapatite.Sodiumdodecyl crosomal activities, these authorssuggestedthe likely identity sulfate-polyacrylamidegel electrophoresis revealed anof the neutrophil enzyme and the liver microsomal NADH-. The NADH inhibitor (Kt= 118 p ~ ) N. o activity was seen with dehydrogenase from neutrophil membranes of beef is distinct.
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