[Purification of pig adrenal 3 beta-hydroxysteroid dehydrogenase/isomerase and influence on microsomal membrane destruction on its enzyme activity].
A 3 beta-hydroxysteroid dehydrogenase/delta 4-delta 5-isomerase (3 beta-HSD/isomerase) has been purified to homogeneity from the pig adrenal microsomes by solubilization with sodium cholate, followed by some conventional column chromatographies. The molecular weight of the enzyme was estimated to be 42000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Km value for pregnenolone as the substrate of the purified enzyme was higher than that of the microsomal enzyme. The purified enzyme was less stable for heat treatment than the microsomal binding enzyme. The microsomal enzyme was treated with various agents or enzymes which destroyed the membrane structure. Lipid peroxidation with Fe2+, digestion with phospholipase A2 or C, and the addition of various free fatty acids, imidazole containing compounds and polymyxin B were utilized for the membrane destruction. Consequently, the 3 beta-HSD/isomerase activity was significantly reduced in all cases, and the Km value of the 3 beta-HSD/isomerase for pregnenolone increased. And the Vmax and Vmax/Km values significantly decreased. Therefore, it is strongly suggested that the normal membrane structure plays an important role in the maintenance of the 3 beta-HSD/isomerase activity.
Nakajin
S
S
Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo, Japan.
Shin
H
H
Shinoda
M
M
D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D000311:Adrenal Glands / Q000166:cytology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology*; D013552:Swine
D000975:Antioxidants; D005230:Fatty Acids, Nonesterified; D007093:Imidazoles; C029899:imidazole; D015096:3-Hydroxysteroid Dehydrogenases