A substantial proportion of cardiac G s is not associated with the plasma membrane

Abstract

The precise interactions between the subunits of G s, (α, β, γ) and the plasma membrane remain to be established. If α s is associated loosely with the inner membrane, is labile during activation, or is always present to some extent in the cytoplasm, then it should fractionate to the supernatant of a high-speed centrifugation. We identified abundant α, (52–66% of total cellular) in the supernatant fraction of right atrial and left ventricular membrane preparations of porcine heart as shown by two distinct measures of α, (immunoblotting and ADP ribosylation by cholera toxin). However, functional assays utilizing reconstitution of cardiac α, with cyc S49 membranes revealed that the supernatant fraction contained ∼16% of total cellular α s activity. The α s present in the supernatant fraction did not result from contamination by sarcolemmal membrane fragments. We conclude that traditional methods for quantifying α, which utilize only detergent extracts from high-speed pellets do not account for a sizable proportion of total cellular α s but that the majority of this population of cardiac α s may not be functional, at least with respect to adenylyl cyclase activation.