NAD(-DEPENDENT ADP-RIBOSYLATION OF T-LYMPHOCYTE ALLOANTIGEN RT6.1 REVERSIBLY PROCEEDING IN INTACT RAT LYMPHOCYTES())

Rat T lymphocyte alloantigen 6.1 (RT6.1), which was synthesized as the fusion protein with a maltose-binding protein in Escherichia coli, di splayed NAD(+)-dependent auto-ADP-ribosylation in addition to an enzym e activity of NAD(+) glycohydrolase. Such ADP-ribosylation of RT6.1 wa s also observed in lymphocytes isolated from rat tissues as follows, W hen intact rat lymphocytes expressing RT6.1 mRNA were incubated with [ alpha-P-32]NAD(+), its radioactivity was incorporated into a cell surf ace protein with the M(r) of 31,000, The radiolabeled 31-kDa protein w as released from the cell surface by treatment of the cells with phosp hatidylinositol-specific phospholipase C and immunoprecipitated with a nti-RT6.1 antiserum, The radioactivity incorporated into the 81-kDa pr otein was recovered as 5'-[P-32]AMP upon incubation with snake venom p hosphodiesterase and also removed by NH2OH treatment, These results su ggested that the NAD(+)-dependent modification of the 31-kDa protein w as due to ADP-ribosylation of glycosylphosphatidylinositol-anchored RT 6.1 at an arginine residue, When intact lymphocytes, in which RT6.1 ha d been once modified by [P-32]ADP-ribosylation, were further incubated in the absence of NAD(+), there was reduction of the radioactivity in the [P-32]ADP-ribosylated RT6.1, The reduced radioactivity was recove red from the incubation medium as [P-32]ADP-ribose. This reduction was effectively inhibited by the addition of ADP-ribose to the reaction m ixture, Moreover, readdition of NAD(+) caused the ADP-ribosylation of RT6.1 again, Thus, the ADP-ribosylation of RT6.1 appeared to proceed r eversibly in intact rat lymphocytes.