Modulatory role of GTP-binding proteins in the endogenous ADP- ribosylation of cytosolic proteins

Abstract

The endogenous ADP-ribosylation of cytosolic proteins and the pattern of NAD degradation were analyzed in subcellular fractions of rat liver in order to investigate the modulation of these reactions by GTP-binding (G) proteins. We could show that intracellular membranes from rat liver have a guanine nucleotide- and divalent cation-dependent pyrophosphatase activity able to rapidly degrade NAD to AMP. This enzymatic activity was investigated by two different approaches: the degradation of [32P]-NAD in the presence of intracellular membranes and the mono-ADP-ribosylation of cytosolic proteins. Divalent cations, preferentially Zn2+ and Mn2+, were required for the pyrophosphatase activity, since in the presence of the Zn2+ chelator TPEN (N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine) or EDTA, the NAD degradation was inhibited by about 50%. Accordingly, in the presence of TPEN the endogenous ADP-ribosylation of cytosolic proteins was enhanced, whereas Zn2+ caused a significant inhibition of this reaction. GDPβS was able to strongly activate the mono-ADP-ribosylation of cytosolic proteins. This effect was abolished by GTPγS, suggesting that a G protein, or rather one of the subunits of a heterotrimeric G protein, is involved in the modulation of the pyrophosphatase and consequently, of endogenous ADP-ribosylation. We propose that a regulatory pathway involving a heterotrimeric G protein modulates enzymes affecting the NAD turnover and availability of NAD for endogenous mADPRTs.