Microsomal 5-ane-3 beta-hydroxysteroid oxidoreductase from pubertal rat Leydig cells: partial purification and characterization.
A 3 beta-hydroxysteroid oxidoreductase which acts on 5 alpha (beta)-reduced C19 and C21 steroids (5-ane-3 beta-hydroxysteroid oxidoreductase; 5-ane-3 beta-HSO) has been solubilized from pubertal rat Leydig cell microsomes and purified 300-fold by ion exchange and gel filtration chromatography. The partially purified enzyme is stable only in the presence of 0.4 M NaCl and appears to exist as a molecule having a molecular weight of 35,000 or as aggregates with a molecular weight in excess of 150,000. NAD+ and NADH+ are used exclusively as cofactors. The velocity of the steroid oxidation reaction was unaffected by either Ca2+ or Mg2+. The steroid oxidation reaction has a pH optimum between 8.0 and 8.5, a temperature optimum at 35 degrees C and an activation energy of 12,850 cal/mol. The pH optimum of the steroid reduction reaction is 6.6. A variety of 5 alpha-reduced C19 and C21 steroids can be utilized as substrates. Treatment of microsomes with phospholipase A2 resulted in a 26 to 90% loss of enzyme activity, paralleling decreased microsomal phospholipid content, and suggesting a role for phospholipids in 5-ane-3 beta-HSO activity. Assays with combined substrates indicate that one enzyme is responsible for activities observed with 5 alpha- and 5 beta-reduced C19- and 5 alpha-reduced C21-3 beta-hydroxysteroids. Purification data indicate that the 5-ane-3 beta-HSO and the 5-ene-3 beta-hydroxysteroid oxidoreductase:isomerase are distinct enzymes.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D007526:Isoelectric Point; D007985:Leydig Cells / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000201:enzymology; D051381:Rats; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature
D006913:Hydroxysteroid Dehydrogenases; D008274:Magnesium; D002118:Calcium