Isolation of a cytochrome P-450 that catalyzes the 25-hydroxylation of vitamin D3 from rat liver microsomes

Shin Ichi Hayashi, Mitsuhide Noshiro, Kyuichiro Okuda

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

A molecular species of cytochrome P-450 that catalyzes the 25-hydroxylation of cholecalciferol (P-450cc25) was purified from rat liver microsomes on the basis of its catalytic activity. The purification procedure consisted of polyethylene glycol fractionation, and column chromatographies on octylamino Sepharose 4B, hydroxylapatite, DEAE-Sepharose CL-6B, and CM-Sepharose CL-6B. The specific cytochrome P-450 content of the final preparation was 17.0 nmol/mg of protein. The enzymatic activity was reconstituted with the purified cytochrome P-450, NADPHcytochrome P-450 reductase, an NADPH-generating system, and dilauroylglyceryl- 3-phosphorylcholine, the specific activity obtained being 3.7 nmol/min/mg of protein, which was 4,000 times as high as that in microsomes. The apparent molecular weight of the P-450cc25 was 50,000, based on the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis. The absorption spectra of the oxidized form of the enzyme showed a Soret band at 416 nm, which is typical of the low spin state of cytochrome P-450, and α and β bands at 570 and 536 nm, respectively. The Soret peak of the reduced cytochrome P-450-CO complex was at 450 nm. The purified enzyme not only catalyzed the 25-hydroxylation of cholecalciferol but also showed hydroxylation activity toward a variety of substrates, i.e. lα-hydroxycholecalciferol (at 25), testosterone (at 2α and 16α) and dehydroepiandrosterone (at 16α). Amino terminal sequence of the purified cytochrome P-450 was determined by the manual sequence method to be H2N-Met-Asp-Pro-Val-Leu-Val-Leu-Val-. The antibody elicited against the purified enzyme in a rabbit inhibited the cholecalciferol 25-hydroxylation activity by more than 90% with a concentration of 2 mg of immunoglubulin per nmol of cytochrome P-450.

Original languageEnglish
Pages (from-to)1753-1763
Number of pages11
JournalJournal of biochemistry
Volume99
Issue number6
DOIs
Publication statusPublished - 1986 Jan 1
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'Isolation of a cytochrome P-450 that catalyzes the 25-hydroxylation of vitamin D3 from rat liver microsomes'. Together they form a unique fingerprint.

  • Cite this