Oxidation-reduction components of a reduced diphosphopyridine nucleotide dehydrogenase

Kumar, S. A. ; Appaji Rao, N. ; Felton, S. P. ; Huennekens, F. M. ; Mackler, Bruce (1968) Oxidation-reduction components of a reduced diphosphopyridine nucleotide dehydrogenase Archives of Biochemistry and Biophysics, 125 (2). pp. 436-448. ISSN 0003-9861

Full text not available from this repository.

Official URL: http://linkinghub.elsevier.com/retrieve/pii/000398...

Related URL: http://dx.doi.org/10.1016/0003-9861(68)90600-0


DPNH dehydrogenase, solubilized from a particulate DPNH oxidase by treatment of the latter with ethanol at pH 4.8 and 43°, has been further purified by precipitation with ammonium sulfate at pH 7.8 and by filtration through Sephadex G-25. The enzyme migrates as a single band when subjected to electrophoresis on starch gel or polyacrylamide. Based upon a molecular weight of 70,000 (determined by filtration through Sephadex G-200), each molecule of protein contains 1 FMN, 2 nonheme irons, 1 labile sulfide group, and 7 thiol groups. The latter are determined by titration of the protein with DTNB in the presence of 4 M urea. When the enzyme is reduced by DPNH under anaerobic conditions and in the absence of an external electron acceptor, the flavin appears to be fully reduced rather than at the semiquinone level; under these conditions, each mole of enzyme oxidizes approximately 3 moles of DPNH. Removal of the bound FMN by passage of the enzyme through Florisil or Bio-Gel does not affect the DPNH-ferricyanide activity while completely abolishing activity with cytochrome c or 2,6-dichlorophenolindophenol; cytochrome c activity can be largely restored by the addition of FMN. p-Chloromercuribenzoate (pCMB) inhibits the dehydrogenation of DPNH coupled to ferricyanide, indophenol, or cytochrome c; inhibition of the DPNH-ferricyanide activity is a biphasic function of pCMB concentration. Preincubation of the enzyme with DPNH increases the sensitivity toward pCMB when ferricyanide and cytochrome c are used as acceptors.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
ID Code:21228
Deposited On:20 Nov 2010 09:17
Last Modified:12 May 2011 12:31

Repository Staff Only: item control page