PURIFICATION AND PROPERTIES OF a Β-Hydroxysteroid DEHYDROGENASE

Talalay, Paul; Dobson, Marie Mollomo

doi:10.1016/s0021-9258(18)49226-5

Cited by 91 publications

(4 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3a-HSD is not the first steroid-transforming enzyme shown to bind steroid ligands backward. For example, 3(17)0hydroxysteroid dehydrogenase from Pseudomonas testosteroni catalyzes the reversible oxidation of dehydroepiandrosterone (DHEA, 30-hydroxyandrost-5-en-17-one) and testosterone (170-hydroxyandrost-4-en-3-one) (Talalay & Dobson, 1953), and these steroids contain the reactive alcohol at either the C-3 or the C-17 position, respectively. Further, it was shown by using a single steroid substrate, e.g., DHEA, that sequential reduction of the 17-oxo group and oxidation of the 3-hydroxy group occurred without dissociation of cofactor (Minard et al, 1985).…”

Section: Discussionmentioning

confidence: 99%

Secosteroid Mechanism-Based Inactivators and Site-Directed Mutagenesis as Probes for Steroid Hormone Recognition by 3.alpha.-Hydroxysteroid Dehydrogenase

Schlegel

Pawlowski²,

Hu³

et al. 1994

Biochemistry

View full text Add to dashboard Cite

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) inactivates circulating androgens, progestins, and glucocorticoids. 3 alpha-HSD is a member of the aldo-keto reductase superfamily, and the X-ray structure of the apoenzyme shows the presence of an (alpha/beta)8 barrel [Hoog, S. S., Pawlowski, J. E., Alzari, P. M., Penning, T. M., & Lewis, M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2517-2521]. As yet, a three-dimensional structure of the ternary complex E.NADPH.steroid is unavailable. To identify regions of the enzyme involved in steroid hormone recognition, we have employed mechanism-based inactivators and site-directed mutagenesis. (3 RS)-1,10-Seco-5 alpha-estr-1-yne-3,17 beta-diol (1) and (17 RS)- 17-hydroxy-14,15-secoandrost-4-en-15-yn-3-one (3) are secosteroids which contain latent Michael acceptors (alpha,beta-unsaturated alcohols) at opposite ends of the steroid nucleus (at the C-3 and C-17 positions, respectively). It was found that compounds 1 and 3 inactivated 3 alpha-HSD only in the presence of NAD+. The requirement for cofactor implies that 1 and 3 are oxidized to the corresponding alpha,beta-unsaturated ketones for inactivation to occur. Chemically prepared 17 beta-hydroxy-1,10-seco-5 alpha-estr-1-yn-3-one (2) and 14,15-secoandrost-4-en-15-yne-3,17-dione (4), the presumed products of 1 and 3 oxidation, behaved as stoichiometric inactivators of 3 alpha-HSD. In the presence and absence of NAD+, 2 and 4 inactivated > 50% of the enzyme in 10 s or less.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

Section: Discussionmentioning

confidence: 99%

Secosteroid Mechanism-Based Inactivators and Site-Directed Mutagenesis as Probes for Steroid Hormone Recognition by 3.alpha.-Hydroxysteroid Dehydrogenase

Schlegel

Pawlowski²,

Hu³

et al. 1994

Biochemistry

View full text Add to dashboard Cite

show abstract

“…The synthesis was performed enzymatically by employing 3/3hydroxysteroid dehydrogenase (33-HSD) from Pseudomonas testosteroni. This enzyme, in the presence of ß-NAD, converts the A5-33-hydroxy group of a variety of steroids to a A4-3-keto group (Talalay & Dobson, 1953). Briefly, 100 nmol of 33,163-dihydroxyandrost-5-en-17-one was dissolved in 0.1 M sodium phosphate, pH 8.9, containing 0.1 unit/mL 33-HSD.…”

Section: Methodsmentioning

confidence: 99%

Multiple sites of steroid hydroxylation by the liver microsomal cytochrome P-450 system: primary and secondary metabolism of androstenedione

Sheets¹,

Estabrook²

1985

Biochemistry

View full text Add to dashboard Cite

To investigate the potential interaction of the various pathways of androgen hydroxylation, we have conducted studies to identify the profile of products formed during the time course of metabolism of androst-4-ene-3,17-dione (AD). Incubates containing AD, NADPH, and liver microsomes (from rats pretreated with phenobarbital) were sampled at times between 0 and 20 min and the metabolites resolved by reverse-phase (C18) high-performance liquid chromatography. By this method, the pattern of formation and of utilization of eight major primary and secondary metabolites of AD was determined. We report here the formation of two previously unidentified major metabolites of AD: 6 beta,16 alpha-dihydroxyandrost-4-ene-3,17-dione and 6 beta,16 beta-dihydroxyandrost-4-ene-3,17-dione. We propose that liver microsomal cytochromes P-450 can sequentially hydroxylate a single molecule of AD at multiple sites. These hydroxylase activities are presumably a result of multiple cytochrome P-450 isozymes acting on AD resulting in a transient time course for the appearance of some monohydroxylated metabolites. In addition, a unidirectional conversion of the metabolite 16 alpha-hydroxyandrost-4-ene-3,17-dione to 16 beta-hydroxyandrost-4-ene-3,17-dione is described. Evidence is provided to support the role of cytochrome P-450 in catalyzing this reaction.

show abstract

“…99, 292) has been in use for some time for the identification and quantitative estimation of the relatively large amounts of 17-ketosteroids in the urine, but partition chromatography is rapidly gaining favor for qualitative (341,353,401,292,257,108,120,217) and quantitative (41,340,211,99,323) analysis. It is a valuable tool for the study of the biosynthesis (56,15,117,354) and tissue metabolism (238,329,450,451,449,223) of androgens, and for the study of enzymatic reactions (414,415,114). Ketosteroids have been identified by paper chromatography in blood (442,271), in adrenal perfusates (42), and partially identified in sperm (113).…”

Section: B Quantitative Methodsmentioning

confidence: 99%

Partition Chromatography Of Steroids

Heftmanń¹

1955

Chem. Rev.

View full text Add to dashboard Cite

PURIFICATION AND PROPERTIES OF a Β-Hydroxysteroid DEHYDROGENASE

Cited by 91 publications

References 20 publications

Secosteroid Mechanism-Based Inactivators and Site-Directed Mutagenesis as Probes for Steroid Hormone Recognition by 3.alpha.-Hydroxysteroid Dehydrogenase

Secosteroid Mechanism-Based Inactivators and Site-Directed Mutagenesis as Probes for Steroid Hormone Recognition by 3.alpha.-Hydroxysteroid Dehydrogenase

Multiple sites of steroid hydroxylation by the liver microsomal cytochrome P-450 system: primary and secondary metabolism of androstenedione

Partition Chromatography Of Steroids

Contact Info

Product

Resources

About