Purification and properties of human hepatic 3 alpha-hydroxysteroid dehydrogenase
| Autor: | A L Southren, Gary G. Gordon, R B Iyer, Ira Schwartz, J M Binstock, Bernard I. Weinstein |
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| Rok vydání: | 1992 |
| Předmět: |
3-Hydroxysteroid Dehydrogenases
Endocrinology Diabetes and Metabolism Clinical Biochemistry Dehydrogenase Eye Biochemistry Substrate Specificity chemistry.chemical_compound Endocrinology Cytosol Humans Isoelectric Point Sodium dodecyl sulfate Molecular Biology Polyacrylamide gel electrophoresis Ammonium sulfate precipitation Chromatography biology Substrate (chemistry) Antibodies Monoclonal Cell Biology Enzyme assay Molecular Weight Kinetics Isoelectric point chemistry Liver Sephadex biology.protein Molecular Medicine |
| Zdroj: | The Journal of steroid biochemistry and molecular biology. 43(4) |
| ISSN: | 0960-0760 |
| Popis: | 3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) was purified greater than 500-fold from human liver cytosol. The purification was monitored using 5 beta-[3H]dihydrocortisol (5 beta-DHF) as substrate. Electrophoretically homogeneous enzyme was obtained using a procedure that involved ammonium sulfate precipitation and three successive column chromatography steps: DEAE-cellulose, hydroxylapatite and Blue-Sepharose. The enzyme is a monomer since the native molecular weight was found to be 37,000, using a calibrated Sephadex G-75 column, and the denatured subunit molecular weight was determined to be 38,500, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The enzyme had a pI of 5.6-5.9. The 3-ketosteroids: cortisol, testosterone, progesterone and androstenedione, were not substrates for 3 alpha-HSD indicating that a saturated 4,5 double bond was required for substrate activity. The conformation at the 5 position, however, did not influence substrate activity since 5 alpha- and 5 beta-DHF and 5 alpha-dihydrotestosterone were all reduced at similar rates. The purified enzyme preferred NADPH to NADH as a cofactor and showed a broad peak of activity in the pH range of 6.8-7.4. The apparent Km for 5 beta-DHF was 18 microM. The enzyme was markedly stabilized by 50 mM phosphate buffer containing 10 to 20% glycerol at 4 degrees C. Freezing and thawing of the enzyme resulted in a large loss of activity during early stages of the purification. This is the first report of the purification to homogeneity of 3 alpha-HSD from human tissue. |
| Databáze: | OpenAIRE |
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