Effects of guanine nucleotides on cholera toxin catalyzed ADP-ribosylation in rat adipocyte plasma membranes

Abstract

ADP-ribosylation of rat adipocyte plasma membrane proteins was investigated following incubation of membranes with [.alpha.-32P]NAD and cholera toxin in the presence and absence of various guanine nucleotides. In membranes incubated without guanine nucleotides, cholera toxin induced incorporation of 32P into 3 discrete proteins of 48, 45 and 41 kDa [kilodaltons]. In membranes containing 100 .mu.M GTP or GDP, toxin-catalyzed incorporation of 32P into the 41 kDa protein was inhibited. GMP and Gpp(NH)p (100 .mu.M) allowed moderate incorporation of 32P into the 41 kDa protein. Toxin-catalyzed labeling of all proteins was rapid, reaching maximal levels between 5-10 min. Toxin-catalyzed ADP-ribosylation of the 48 and 45 kDa proteins was stimulated by GTP, reaching maximal levels at 10-5 M GTP. Inhibition of toxin-dependent labeling of the 41 kDa protein required GTP concentrations > 10-7 M with complete inhibition occurring between 10-5-10-4 M GTP. Cholera toxin catalyzed ADP-ribosylation was increased up to 2-fold in membranes supplemented with adipocyte cytosol. Thus, cholera toxin catalyzes ADP-ribosylation of 3 distinct adipocyte plasma membrane proteins, each of which is regulated by the amount and type of added guanine nucleotides.