Modulatory role of GTP-binding proteins in the endogenous ADP-ribosylation of cytosolic proteins.
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. GDP beta S was able to strongly activate the mono-ADP-ribosylation of cytosolic proteins. This effect was abolished by GTP gamma S, 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.
Di Girolamo
M
M
Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Santa Maria Imbaro (Chieti), Italy.
Lupi
R
R
Silletta
M G
MG
Turacchio
S
S
Iurisci
C
C
Luini
A
A
Corda
D
D
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D003600:Cytosol; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011755:Pyrophosphatases / Q000378:metabolism; D051381:Rats
D011506:Proteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D009244:NAD+ Nucleosidase; D019204:GTP-Binding Proteins; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase