Quantification of the α and β subunits of the transducing elements (Gs and Gi) of adenylate cyclase in adipocyte membranes from lean and obese (ob/ob) mice

Abstract

The abundance of the alpha and beta subunits of the GTP-binding proteins (G-proteins) that transduce hormonal messages to adenylate cyclase was assessed in adipocyte membranes from lean (+/+) and obese (ob/ob) mice, using ADP-ribosylation with bacterial toxin and immunodetection. Both methods revealed two Gs alpha species (48 and 42 kDa) in the membranes. Compared with those of lean mice, the membranes from obese mice contained substantially less of the 48 kDa species of Gs alpha, as assessed by both methods. ADP-ribosylation by pertussis toxin showed that only half as much ADP-ribose was incorporated into Gi alpha in the membranes from obese as compared with lean mice. Immunodetection revealed two separate Gi alpha peptides (39 and 40 kDa) and showed that the 40 kDa species was less abundant in the membranes from obese mice, whereas the amount of the 39 kDa species was similar in membranes from both lean and obese animals. Based on ADP-ribosylation assays, in membranes from lean mice the ratio Gs alpha/Gi alpha was 1:16, whereas in the membranes from obese mice it was 1:10. Similar amounts of immunodetectable beta peptide were found in both types of membranes. On the basis of the currently accepted dissociation model of adenylate cyclase activation, the decrease in the abundance of the Gi alpha subunit in adipocyte membranes from obese mice could account for the abnormal kinetics of the enzyme in these membranes.