Purification and Characterization of the Multiple Forms of 3α-Hydroxysteroid Dehydrogenase in Rat Liver Cytosol

Ikeda, Mikiko; Hattori, Hiroko; Ikeda, Nobuko; Hayakawa, Shohei; Ohmori, Shingo

doi:10.1515/bchm2.1984.365.1.377

Cited by 17 publications

(16 citation statements)

References 13 publications

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“…Furthermore, the enzyme exhibited a marked preference for pyridine nucleotides, especially NADPH. This preference for NADPH has been noted by other investigators (21,22). Since NADPH is the predominant form under normal redox conditions and the reductase activity is optimal at normal intracellular pH, we predicted that the enzyme would function mainly as a reductase in cells.…”

Section: Discussionsupporting

confidence: 78%

“…To further prove that the Y' binders are 3a-hydroxysteroid dehydrogenases, we demonstrated the re- action of binder-I antisera with an identical molecular weight form of 3a-hydroxysteroid dehydrogenase in peaks I, II, and III from chromatofocusing and the homogeneous 3a-hydroxysteroid dehydrogenase-I. Other investigators also have noted multiple molecular forms of 3a-hydroxysteroid dehydrogenase (21).…”

Section: Discussionmentioning

confidence: 69%

“…The cofactor preference ofthese two different enzymes is probably responsible for the epimerization at the 3 position. NAD+ predominates over NADH and is the known preferred cofactor for the microsomal enzyme (25 (9,21,22). The enzyme has been implicated in the biosynthetic oxidoreduction of the 3-position of a wide variety of steroid hormone substrates.…”

Section: Discussionmentioning

confidence: 99%

“…The Km for cofactor preference and pH optimum were similar to our results. More recently, Ikeda et al identified multiple forms of 3a-hydroxysteroid dehydrogenase in rat liver and purified two forms (21,33). Their purification scheme consisted of sequential ammonium sulfate precipitation, anionic and cationic ion exchange chromatography, chromatofocusing, and red-sepharose affinity chromatography.…”

Section: Discussionmentioning

confidence: 99%

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3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance.

Stolz¹,

Takikawa²,

Sugiyama³

et al. 1987

J. Clin. Invest.

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Rat Y' bile acid binders (33 kD) have been previously recognized as cytosolic bile acid binding proteins (Sugiyama, Y., T. Yamada, and N. Kaplowitz, 1983, J. Biol. Chem., 258:3602-3607). We have now determined that these Y' binders are 3a-hydroxysteroid dehydrogenases (3a-HSD), bile acid-metabolizing enzymes. 3a-HSD activity copurified with lithocholic acid-binding activity after sequential gel filtration, chromatofocusing, and affinity chromatography. Three peaks of 3a-HSD activity (I, II, III) were observed in chromatofocusing and all were identified on Western blot by a specific Y' binder antiserum. 3a-HSD-I, the predominant form, was purified and functioned best as a reductase at pH 7.0 with a marked preference for NADPH. Michaelis constant values for mono-and dihydroxy bile acids were 1-2

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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance.

Stolz¹,

Takikawa²,

Sugiyama³

et al. 1987

J. Clin. Invest.

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“…The enzyme 3α-HSD is found in many ocular (Iyer et al, 1993) and extra-ocular tissues and can reduce a variety of substrates such as 3-ketosteroids (Tomkins G. M., 1956 ;Gustafsson et al, 1983), bile acids (Takikawa et al, 1990), prostaglandins (Penning and Sharp, 1987) and xenobiotics (Smithgall et al, 1986). Hepatic 3α-HSD has been purified from many species (Ikeda et al, 1984 ;Penning et al, 1984 ;Worner and Oesch, 1984 ;Hara et al, 1988 ;Morikawa and Amaoki, 1990) including the human (Kudo et al, 1990 ;Iyer et al, 1992 ;Takikawa et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

Decreased 3 α-Hydroxysteroid Dehydrogenase Activity in Peripheral Blood Lymphocytes from Patients with Primary Open Angle Glaucoma

et al. 1996

Experimental Eye Research

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Purification of 3α-hydroxysteroid and 3β-hydroxysteroid dehydrogenases from human liver cytosol

et al. 1992

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We previously reported that the human Y' bile acid binder, which has higher bile acid binding affinities than rat Y' binders (3 alpha-hydroxysteroid dehydrogenases), has dihydrodiol dehydrogenase activity and is different from 3 alpha-hydroxysteroid dehydrogenases. In this study, 3 alpha-hydroxysteroid dehydrogenases and 3 beta-hydroxysteroid dehydrogenase were purified from human liver, and bile acid binding affinities and enzyme kinetics of the 3 alpha-hydroxysteroid dehydrogenases were studied. On chromatofocusing of pooled Affigel blue fraction of the Y' fraction, three 3 alpha-hydroxysteroid dehydrogenase peaks eluted at pH 6.0, 5.7 and 5.4. These peaks did not bind bile acids, and further purification by hydroxyapatite-high-performance liquid chromatography gave pure 3 alpha-hydroxysteroid dehydrogenases with identical M(r) (36,000) having dihydrodiol dehydrogenase activity. 3 beta-Hydroxysteroid dehydrogenase was eluted together with Y' bile acid binder at pH 7.2 on chromatofocusing and was separated from Y' bile acid binder on hydroxyapatite-high-performance liquid chromatography as a pure protein with M(r) 32,000. The apparent Kms of 3 alpha-hydroxysteroid dehydrogenases were similar to those of rat enzymes. In conclusion, we purified human hepatic 3 alpha-hydroxysteroid dehydrogenases, which have similar characteristics to rat enzymes, but do not bind bile acids or reduce bile acid precursors. These data further support the importance of human bile acid binder in intracellular bile acid transport in the human liver.

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Purification and Characterization of the Multiple Forms of 3α-Hydroxysteroid Dehydrogenase in Rat Liver Cytosol

Cited by 17 publications

References 13 publications

3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance.

3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance.

Decreased 3 α-Hydroxysteroid Dehydrogenase Activity in Peripheral Blood Lymphocytes from Patients with Primary Open Angle Glaucoma

Purification of 3α-hydroxysteroid and 3β-hydroxysteroid dehydrogenases from human liver cytosol

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