The HLB dependency for detergent solubilization of hormonally sensitive adenylate cyclase

✍ Scribed by Storm, D. R. ;Field, S. O. ;Ryan, J.

• Publisher: Wiley (John Wiley & Sons)
• Year: 1976
• Tongue: English
• Weight: 570 KB
• Volume: 4
• Category: Article
• ISSN: 0091-7419
• DOI: 10.1002/jss.400040209

✦ Synopsis

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‐epine‐phrine‐, 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 MgCl~2~, 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 × g, the absence of particulate material in the supernatant when examined by electron microscopy, and inclusion of hormonally sensitive adenylate cylcase 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° C.