Isolation, Purification, and Some Properties of Reduced Nicotinamide Adenine Dinucleotide Phosphate-Cytochrome c2 Reductase from Rhodopseudomonas spheroides

A method has been described for the isolation and purification of NADPH-cytochrome c 2 reductase from light-grown Rhodopseudomonas spheroides . The enzyme is a nonmetalloflavoprotein with flavin adenine dinucleotide as the prosthetic group, and it catalyzes the reduction of R. spheroides cytochrome...

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Bibliographic Details
Published in:The Journal of biological chemistry 1968-07, Vol.243 (13), p.3742-3749
Main Authors: Sabo, D J, Orlando, J A
Format: Article
Language:English
Subjects:
Chromatography, Gel
Chromatography, Ion Exchange
Cytochromes
Electrophoresis, Disc
Flavin-Adenine Dinucleotide - analysis
Glutathione
Hydrogen-Ion Concentration
Kinetics
Methods
Molecular Weight
NADP
Oxidoreductases - analysis
Oxidoreductases - antagonists & inhibitors
Rhodopseudomonas - enzymology
Spectrophotometry
Ultracentrifugation
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Summary:A method has been described for the isolation and purification of NADPH-cytochrome c 2 reductase from light-grown Rhodopseudomonas spheroides . The enzyme is a nonmetalloflavoprotein with flavin adenine dinucleotide as the prosthetic group, and it catalyzes the reduction of R. spheroides cytochrome c 2 , 2,6-dichloroindophenol, and K 3 Fe(CN) 6 with NADPH as the electron donor. It does not reduce R. spheroides cytochrome 553 but reduces Rhodospirillum rubrum cytochrome c 2 and mammalian cytochrome c very slowly. There is no NADH or NADPH oxidase activity, NADP + reductase activity, or transhydrogenase activity associated with the enzyme. The pH optimum was found to be 7.5; and K m values of 3.7 x 10 -5 m , 1.25 x 10 -5 m , and 1.24 x 10 -4 m were calculated for cytochrome c 2 , 2,6-dichloroindophenol, and K 3 Fe(CN) 6 reduction, respectively. NADH functioned as a competitive inhibitor, and the K i was calculated to be 5.5 x 10 -5 m . The enzyme was inhibited by p -chloromercuribenzoate, N -ethylmaleimide, iodoacetate, and thyroxine; and the inhibition by sulfhydryl reagents was reversed by reduced glutathione. Preincubation with NADPH protected the enzyme against the poisoning effects of sulfhydryl reagents but not thyroxine. The behavior of the enzyme on Sephadex G-100 and in the analytical ultracentrifuge indicates the presence of several molecular forms of the enzyme.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)34201-2