Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1984 Nov 1;223(3):697–705. doi: 10.1042/bj2230697

Purification and characterization of two forms of microsomal carbonyl reductase in guinea pig liver.

S Usui, A Hara, T Nakayama, H Sawada
PMCID: PMC1144353  PMID: 6439186

Abstract

Two forms of microsomal carbonyl reductase, solubilized in Triton X-100, were purified to homogeneity from the liver of male guinea pigs, primarily by affinity, DEAE-Sephacel, gel-filtration and hydroxyapatite chromatography. The major form was a tetrameric glycoprotein of single subunits of Mr 32000 and a pI value of 7.0; another minor form was a monomeric protein with Mr 34000 and a pI value of 7.8. The enzymes were immunologically distinct. Although the enzymes showed similar substrate specificity for exogenous aldehydes and ketones and apparently absolute cofactor specificity for NADPH, their specificity for natural carbonyl compounds differed. The major form irreversibly reduced 5 alpha- and 5 beta-dihydrotestosterones, menadione and lauryl aldehyde with low Km values of 10-70 microM, whereas the minor form not only reduced 17-oxosteroids, of which 3 alpha-hydroxy-5 beta-androstan-17-one was the best substrate, but also oxidized 17-hydroxysteroids in the presence of NADP+. The two forms of carbonyl reductase also exhibited different sensitivity to heavy metal ions, dicoumarol, tetramethyleneglutaric acid, phenobarbitone and corticosteroids.

Full text

PDF
697

Images in this article

701Fig. 2.
on p.701
703Fig. 4.
on p.703

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ackrell B. A., Kearney E. B., Singer T. P. Mammalian succinate dehydrogenase. Methods Enzymol. 1978;53:466–483. doi: 10.1016/s0076-6879(78)53050-4. [DOI] [PubMed] [Google Scholar]
  2. BEERS R. F., Jr, SIZER I. W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem. 1952 Mar;195(1):133–140. [PubMed] [Google Scholar]
  3. Billheimer J. T., Alcorn M., Gaylor J. L. Solubilization and partial purification of a microsomal 3-ketosteroid reductase of cholesterol biosynthesis. Arch Biochem Biophys. 1981 Oct 1;211(1):430–438. doi: 10.1016/0003-9861(81)90474-4. [DOI] [PubMed] [Google Scholar]
  4. Blomquist C. H., Kotts C. E., Hakanson E. Y. Microsomal 17beta-hydroxysteroid dehydrogenase of guinea pig liver: detergent solubilization and a comparison of kinetic and stability properties of bound and solubilized forms. J Steroid Biochem. 1977 Mar;8(3):193–198. doi: 10.1016/0022-4731(77)90050-4. [DOI] [PubMed] [Google Scholar]
  5. CHUNG A. E., LANGDON R. G. Human erythrocyte glucose 6-phosphate dehydrogenase. I. Isolation and properties of the enzyme. J Biol Chem. 1963 Jul;238:2309–2316. [PubMed] [Google Scholar]
  6. Clarke S. The size and detergent binding of membrane proteins. J Biol Chem. 1975 Jul 25;250(14):5459–5469. [PubMed] [Google Scholar]
  7. Culp H. W., McMahon R. E. Reductase for aromatic aldehydes and ketones. The partial purification and properties of a reduced triphosphopyridine nucleotide-dependent reductase from rabbit kidney cortex. J Biol Chem. 1968 Feb 25;243(4):848–852. [PubMed] [Google Scholar]
  8. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  9. Daly A. K., Mantle T. J. Purification and characterization of the multiple forms of aldehyde reductase in ox kidney. Biochem J. 1982 Aug 1;205(2):373–380. doi: 10.1042/bj2050373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Felsted R. L., Bachur N. R. Mammalian carbonyl reductases. Drug Metab Rev. 1980;11(1):1–60. doi: 10.3109/03602538008994021. [DOI] [PubMed] [Google Scholar]
  11. Felsted R. L., Richter D. R., Jones D. M., Bachur N. R. Isolation and characterization of rabbit liver xenobiotic carbonyl reductases. Biochem Pharmacol. 1980 Jun 1;29(11):1503–1516. doi: 10.1016/0006-2952(80)90601-2. [DOI] [PubMed] [Google Scholar]
  12. Hara A., Deyashiki Y., Nakagawa M., Nakayama T., Sawada H. Isolation of proteins with carbonyl reductase activity and prostaglandin-9-ketoreductase activity from chicken kidney. J Biochem. 1982 Dec;92(6):1753–1762. doi: 10.1093/oxfordjournals.jbchem.a134105. [DOI] [PubMed] [Google Scholar]
  13. Ikeda M., Ezaki M., Kokeguchi S., Ohmori S. Studies on NADPH-dependent chloral hydrate reducing enzymes in rat liver cytosol. Biochem Pharmacol. 1981 Jul 15;30(14):1931–1939. doi: 10.1016/0006-2952(81)90202-1. [DOI] [PubMed] [Google Scholar]
  14. Kahl G. F. Experiments on the metyrapone reducing microsomal enzyme system. Naunyn Schmiedebergs Arch Pharmakol. 1970;266(1):61–74. doi: 10.1007/BF00997783. [DOI] [PubMed] [Google Scholar]
  15. Kawalek J. C., Gilbertson J. R. Enzymic reduction of free fatty aldehydes in bovine cardiac muscle. Biochem Biophys Res Commun. 1973 Apr 16;51(4):1027–1033. doi: 10.1016/0006-291x(73)90030-2. [DOI] [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. Mather I. H., Tamplin C. B. A method for the determination of protein in the presence of Triton X-100. Anal Biochem. 1979 Feb;93(1):139–142. [PubMed] [Google Scholar]
  18. Nakayama T., Hara A., Sawada H. Purification and characterization of a novel pyrazole-sensitive carbonyl reductase in guinea pig lung. Arch Biochem Biophys. 1982 Sep;217(2):564–573. doi: 10.1016/0003-9861(82)90538-0. [DOI] [PubMed] [Google Scholar]
  19. RACKER E. Crystalline alcohol dehydrogenase from baker's yeast. J Biol Chem. 1950 May;184(1):313–319. [PubMed] [Google Scholar]
  20. Sawada H., Hara A., Hayashibara M., Nakayama T. Guinea pig liver aromatic aldehyde-ketone reductases identical with 17 beta-hydroxysteroid dehydrogenase isozymes. J Biochem. 1979 Oct;86(4):883–892. doi: 10.1093/oxfordjournals.jbchem.a132620. [DOI] [PubMed] [Google Scholar]
  21. Sawada H., Hara A., Hayashibara M., Nakayama T., Usui S., Saeki T. Microsomal reductase for aromatic aldehydes and ketones in guinea pig liver. Purification, characterization, and functional relationship to hexose-6-phosphate dehydrogenase. J Biochem. 1981 Oct;90(4):1077–1085. doi: 10.1093/oxfordjournals.jbchem.a133559. [DOI] [PubMed] [Google Scholar]
  22. Sawada H., Hara A., Kato F., Nakayama T. Purification and properties of reductases for aromatic aldehydes and ketones from guinea pig liver. J Biochem. 1979 Oct;86(4):871–881. doi: 10.1093/oxfordjournals.jbchem.a132619. [DOI] [PubMed] [Google Scholar]
  23. Sawada H., Hara A., Nakayama T., Kato F. Reductases for aromatic aldehydes and ketones from rabbit liver. Purification and characterization. J Biochem. 1980 Apr;87(4):1153–1165. [PubMed] [Google Scholar]
  24. Sawada H., Hara A. Studies on metabolism of bromazepam. VI. Reduction of 2-(2-amino-5-bromobenzoyl)pyridine, a metabolite of bromazepam, in the rabbit, rat, and guinea pig. Drug Metab Dispos. 1978 May-Jun;6(3):205–212. [PubMed] [Google Scholar]
  25. Sawada H., Sasaki E., Asano S., Hara A. [Studies on human prostatic acid phosphatase. I. Effect of detergent and bovine serum albumin on hydrolysis of various substrates by purified prostatic acid phosphatase (author's transl)]. Yakugaku Zasshi. 1978 Sep;98(9):1167–1172. doi: 10.1248/yakushi1947.98.9_1167. [DOI] [PubMed] [Google Scholar]
  26. WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]
  27. Wallin R., Hutson S. Vitamin K-dependent carboxylation. Evidence that at least two microsomal dehydrogenases reduce vitamin K1 to support carboxylation. J Biol Chem. 1982 Feb 25;257(4):1583–1586. [PubMed] [Google Scholar]
  28. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  29. Wermuth B. Purification and properties of an NADPH-dependent carbonyl reductase from human brain. Relationship to prostaglandin 9-ketoreductase and xenobiotic ketone reductase. J Biol Chem. 1981 Feb 10;256(3):1206–1213. [PubMed] [Google Scholar]
  30. Zacharius R. M., Zell T. E., Morrison J. H., Woodlock J. J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem. 1969 Jul;30(1):148–152. doi: 10.1016/0003-2697(69)90383-2. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES