Purification of catalytically active hepatic NADPH cytochrome P450 oxidoreductase from the rhesus monkey, Macaca mulatta

Kota Ramana, Krishan K. Kohli

Research output: Contribution to journalArticle

Abstract

Hepatic NADPH cytochrome P450 oxidoreductase capable of supporting polysubstrate monooxygenase (PSMO) reactions was purified from microsomes obtained from phenobarbitone (PB) pretreated rhesus monkey. Two preparations of the enzyme purified by affinity and molecular exclusion chromatographic techniques demonstrated specific content of 19.5 and 37.9 nmol cytochrome c reduced/min/mg protein and subunit molecular weight of 66 and 80 kDa, respectively. Both forms supported oxidation of NADPH and reduction of cytochrome c and DCIP but only 80 kDa preparation supported PSMO reactions. The reconstituted system consisted of hepatic P450, NADPH cytochrome P450 oxidoreductase, cytochrome b5 all purified from PB pretreated rhesus monkey and dilauroyl phosphatidylcholine or microsomal lipid. Eighty kDa preparation supported the metabolism of aminopyrine and tolbutamide by hepatic P4502C and erythromycin, ethylmorphine and nifedipine by hepatic P450 3A, respectively. The turnover of these substrates increased in the presence of partially purified cytochrome b5 from the rhesus monkey. To best of our knowledge this is the first report on the purification of monkey hepatic NADPH cytochrome P450 oxidoreductase capable of supporting in vitro PSMO by different isozymes of P450.

Original languageEnglish (US)
Pages (from-to)960-966
Number of pages7
JournalIndian Journal of Experimental Biology
Volume36
Issue number10
StatePublished - 1998
Externally publishedYes

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NADPH-Ferrihemoprotein Reductase
Mixed Function Oxygenases
Macaca mulatta
Purification
Cytochromes b5
Phenobarbital
Cytochromes c
Proteins
Liver
Ethylmorphine
Aminopyrine
Tolbutamide
Protein Subunits
Erythromycin
Nifedipine
Phosphatidylcholines
NADP
Metabolism
Isoenzymes
Molecular weight

ASJC Scopus subject areas

  • Bioengineering

Cite this

Purification of catalytically active hepatic NADPH cytochrome P450 oxidoreductase from the rhesus monkey, Macaca mulatta. / Ramana, Kota; Kohli, Krishan K.

In: Indian Journal of Experimental Biology, Vol. 36, No. 10, 1998, p. 960-966.

Research output: Contribution to journalArticle

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