ADP ribosylation of membrane proteins from human fibroblasts. Effect of prior exposure of cells to choleragen

Abstract

Major labeled bands of Mr = 42,000 and 47,000 were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis of human skin fibroblast membranes incubated with choleragen and [32P]NAD. Prior incubation of intact fibroblasts with choleragen blocked specifically the subsequent in vitro labeling of these two proteins. The effect of choleragen was dependent on time, temperature, and toxin concentration. Neither the choleragen A subunit nor the B subunit nor the A1 peptide could replace the holotoxin in cell incubations. Inhibition of subsequent in vitro labeling by prior exposure of cells to choleragen was correlated with increased cellular cAMP. Incubation of fibroblasts with prostaglandin E1 and isoproterenol, which activate adenylate cyclase by different mechanisms, did not block subsequent labeling with choleragen and [32P]NAD. The results suggest that proteins of Mr = 42,000 and 47,000 may be in vivo substrates for choleragen in human fibroblasts.