cDNA sequence and catalytic properties of a chick embryo alcohol dehydrogenase that oxidizes retinol and 3β,5α-hydroxysteroids

Natalia Y. Kedishvili, Wendy H. Cough, Ellen A G Chernoff, Thomas Hurley, Carol L. Stone, Kenneth D. Bowman, Kirill M. Popov, William F. Bosron, Ting Kai Li

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

This study was undertaken to identify the cytosolic 40-kDa zinc- containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues. Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified. The new cDNA hybridized with two messages of ~2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain. The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver λgt11 cDNA library. The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of ~40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min- 1 for ethanol at pH 7.4 and 25 °C. ADH-F was stereospecific for the 3β,5α-versus 3β,5β-hydroxysteroids. The K(m) value for ethanol at pH 7.4 was 17 mM compared with 56/μM for all-transretinal and 31 μM for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.

Original languageEnglish
Pages (from-to)7494-7500
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number11
DOIs
StatePublished - Mar 14 1997

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Hydroxysteroids
Alcohol Dehydrogenase
Chick Embryo
Vitamin A
Complementary DNA
Chickens
Liver
Base Pairing
Ethanol
Androsterone
Limb Buds
DNA Primers
Polymerase chain reaction
Molecular mass
Titration
Gene Library
NAD
Escherichia coli
Open Reading Frames
Isoenzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

cDNA sequence and catalytic properties of a chick embryo alcohol dehydrogenase that oxidizes retinol and 3β,5α-hydroxysteroids. / Kedishvili, Natalia Y.; Cough, Wendy H.; Chernoff, Ellen A G; Hurley, Thomas; Stone, Carol L.; Bowman, Kenneth D.; Popov, Kirill M.; Bosron, William F.; Li, Ting Kai.

In: Journal of Biological Chemistry, Vol. 272, No. 11, 14.03.1997, p. 7494-7500.

Research output: Contribution to journalArticle

Kedishvili, NY, Cough, WH, Chernoff, EAG, Hurley, T, Stone, CL, Bowman, KD, Popov, KM, Bosron, WF & Li, TK 1997, 'cDNA sequence and catalytic properties of a chick embryo alcohol dehydrogenase that oxidizes retinol and 3β,5α-hydroxysteroids', Journal of Biological Chemistry, vol. 272, no. 11, pp. 7494-7500. https://doi.org/10.1074/jbc.272.11.7494
Kedishvili, Natalia Y. ; Cough, Wendy H. ; Chernoff, Ellen A G ; Hurley, Thomas ; Stone, Carol L. ; Bowman, Kenneth D. ; Popov, Kirill M. ; Bosron, William F. ; Li, Ting Kai. / cDNA sequence and catalytic properties of a chick embryo alcohol dehydrogenase that oxidizes retinol and 3β,5α-hydroxysteroids. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 11. pp. 7494-7500.
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AU - Hurley, Thomas

AU - Stone, Carol L.

AU - Bowman, Kenneth D.

AU - Popov, Kirill M.

AU - Bosron, William F.

AU - Li, Ting Kai

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