Expression of a rat liver microsomal cytochrome P-450 catalyzing testosterone 16α-hydroxylation in Saccharomyces cerevisiae: Vitamin D3 25-hydroxylase and testosterone 16α-hydroxylase are distinct forms of cytochrome P-450

Shin Ichi Hayashi, Ken-Ichirou Morohashi, Hidefumi Yoshioka, Kyuichiro Okuda, Tsuneo Omura

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Abstract

Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 × 105 molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16α- and 2α-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or lα-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5β-cholestane-3α, 7α, 12α-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2α- and 16α-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16α-and 2α-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.

Original languageEnglish
Pages (from-to)858-862
Number of pages5
JournalJournal of biochemistry
Volume103
Issue number5
DOIs
Publication statusPublished - Jan 1 1988

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Cholestanetriol 26-Monooxygenase
Hydroxylation
Mixed Function Oxygenases
Liver
Yeast
Cytochrome P-450 Enzyme System
Saccharomyces cerevisiae
Testosterone
Rats
Complementary DNA
Yeasts
Cholecalciferol
Cholestanes
Calcifediol
Genetic Vectors
Hydroxycholecalciferols
Alcohol Dehydrogenase
Cytochromes c
Escherichia coli
Catalyst activity

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

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title = "Expression of a rat liver microsomal cytochrome P-450 catalyzing testosterone 16α-hydroxylation in Saccharomyces cerevisiae: Vitamin D3 25-hydroxylase and testosterone 16α-hydroxylase are distinct forms of cytochrome P-450",
abstract = "Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 × 105 molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16α- and 2α-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or lα-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5β-cholestane-3α, 7α, 12α-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2α- and 16α-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16α-and 2α-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.",
author = "Hayashi, {Shin Ichi} and Ken-Ichirou Morohashi and Hidefumi Yoshioka and Kyuichiro Okuda and Tsuneo Omura",
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T1 - Expression of a rat liver microsomal cytochrome P-450 catalyzing testosterone 16α-hydroxylation in Saccharomyces cerevisiae

T2 - Vitamin D3 25-hydroxylase and testosterone 16α-hydroxylase are distinct forms of cytochrome P-450

AU - Hayashi, Shin Ichi

AU - Morohashi, Ken-Ichirou

AU - Yoshioka, Hidefumi

AU - Okuda, Kyuichiro

AU - Omura, Tsuneo

PY - 1988/1/1

Y1 - 1988/1/1

N2 - Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 × 105 molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16α- and 2α-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or lα-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5β-cholestane-3α, 7α, 12α-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2α- and 16α-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16α-and 2α-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.

AB - Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 × 105 molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16α- and 2α-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or lα-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5β-cholestane-3α, 7α, 12α-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2α- and 16α-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16α-and 2α-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.

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