Nitric oxide stimulates the ADP-ribosylation of actin in human neutrophils

ADP-ribosylation is an important post-translational protein modification; however, endogenous substrates are poorly characterized. In these studies we examined the effects of nitric oxide on the ADP-ribosylation of neutrophil proteins. Purified cytosol and plasma membrane were incubated with 32P-NAD (5 microM, 1 microCi, 30 min) in the presence or absence of nitric oxide. Nitric oxide induced the ADP-ribosylation of the 37 kD substrate present only in cytosol. Nitric oxide treatment of plasma membrane plus cytosol revealed the ADP-ribosylation of an additional 43 kD protein. This 43 kD substrate was identified as actin by both phalloidin precipitation and immunoblot (2-D) gel using specific anti-actin antibodies. The data indicate that nitric oxide stimulates the ADP-ribosylation of two discrete substrates in fractionated PMN, one of which can be identified as actin. NO-induced ADP-ribosylation may contribute to the modulatory effect of nitric oxide on neutrophil functions, including F-actin assembly.