| Abstract: | The HLB dependency for the solubilization of membrane proteins and adenylate cyclase activity from a plasma membrane-enriched fraction from rat liver has been determined. The HLB (hydrophilic/lipophilic/balance) number of a detergent is an empirical measure of its relative hydrophobicity. Detergent HLB numbers vary systematically with the length of the ethylene oxide chain for a homologous series of detergents such as the Triton X series. These detergents have a constant hydrophobic moiety, octylphenyl, and a variable polar portion, polyethoxyethanol. Basal-NaF-epinephrine-, and glucagon-stimulated adenylate cyclase activities were solubilized in the HLB range of 16.8-17.4. Solubilization was most effective in 0.01 M Tris buffers at pH 7.5 containing 1-5 mM mercaptoethanol, 1 mM MgCl2, and 0.1% Triton X-305. The detergent to membrane protein ratio used in these studies was 3:1. Criteria for solubilization included lack of sedimentation at 100,000 X g, the absence of particulate material in the supernatant when examined by electron microscopy, and inclusion of hormonally sensitive adenylate cyclase activity in Sephadex G-200 gels. The apparent molecular weight of the solubilized enzyme was approximately 200,000 in the presence of Triton X-305. The solubilized enzyme was stimulated 5-fold by NaF, 7-fold by glucagon, and 20-fold by epinephrine compared to the particulate enzyme used in this study which was stimulated 10-fold, 3.4-fold, and 4-fold by NaF, epinephrine, and glucagon, respectively. The solubilized enzyme is stable for several weeks when stored at -60 degrees C. |
