Detergent solubilization of 3 beta-hydroxysteroid dehydrogenase from dog pancreas.
Functional 3 beta-hydroxysteroid dehydrogenase coupled with isomerase (3 beta-HSD) was extracted from dog pancreatic mitochondria by treatment with the zwitterionic detergent CHAPSO. Increasing concentrations of this detergent led to a progressive and simultaneous solubilization of the pregnene (C-21) and androstene (C-19) dehydrogenase activities. Optimal solubilization of both C-21 and C-19 3 beta-HSD activities was achieved at a detergent/protein ratio of 0.6 (w/w). One hundred thirty percent of the initial particulate enzyme activities were recovered in the 105,000 g supernatant fluid with a 2.5-fold increase in the enzymatic specific activities. The C-21/C-19 activity ratios were 1.3 for mitochondria and 1.39 for the solubilized preparation. The apparent Km values for steroid substrates were unchanged after solubilization. Treatment of the mitochondrial suspension with sodium deoxycholate, CTAB, Lubrol XW, Brij 58, Emulgen 913 and Triton X-100 markedly decreased the 3 beta-HSD activities as a function of the detergent concentration and failed in to achieve solubilization.
Mendoza-Hernández
G
G
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F., México.
Libreros-Minotta
C A
CA
Rendón
J L
JL
D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002793:Cholic Acids; D003687:Dehydroepiandrosterone / Q000378:metabolism; D003902:Detergents; D004285:Dogs; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D010179:Pancreas / Q000201:enzymology*; D011284:Pregnenolone / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity
D002793:Cholic Acids; D003902:Detergents; D003687:Dehydroepiandrosterone; D011284:Pregnenolone; C048531:chapso; D015096:3-Hydroxysteroid Dehydrogenases