The leucoyte surface antigen CD38 has been shown to be an ecto-enzyme with multiple catalytic activities. It is principally a NAD+ glycohydrolase that transforms NAD+ into ADP-ribose and nicotinamide. CD38 is also able to produce small amounts of cyclic ADP-ribose (ADP-ribosyl cyclase activity) and to hydrolyse this cyclic metabolite into ADP-ribose (cyclic ADP-ribose hydrolase activity). To classify CD38 among the enzymes that transfer the ADP-ribosyl moiety of NAD+ to a variety of acceptors, we have investigated its substrate specificity and some characteristics of its kinetic and molecular mechanisms. We find that CD38-catalysed cleavage of the nicotinamide-ribose bond results in the formation of an E.ADP-ribosyl intermediary complex, which is common to all reaction pathways; this intermediate reacts (1) with acceptors such as water (hydrolysis), methanol (methanolysis) or pyridine (transglycosidation), and (2) intramolecularly, yielding cyclic ADP-ribose with a low efficiency. This reaction scheme is also followed when using nicotinamide guanine dinucleotide as an alternative substrate; in this case, however, the cyclization process is highly favoured. The results obtained here are not compatible with the prevailing model for the mode of action of CD38, according to which this enzyme produces first cyclic ADP-ribose which is then immediately hydrolysed into ADP-ribose (i.e. sequential ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities). We show instead that the cyclic metabolite was a reaction product of CD38 rather than an obligatory reaction intermediate during the glycohydrolase activity. Altogether our results lead to the conclusion that CD38 is an authentic 'classical' NAD(P)+ glycohydrolase (EC 3.2.2.6).

We have shown that bovine spleen NAD ؉ glycohydrolase (EC 3.2.2.5), purified to homogeneity, is a multifunctional enzyme. A time-dependent formation of cADPR from NAD ؉ that did not exceed 1.5-2% of the reaction products was measurable. The cyclase activity of this enzyme was, however, best evidenced by its transformation of NGD ؉ into cyclic GDP-ribose (cGDPR). The formation of the cyclic compound could be monitored spectroscopically (UV and fluorescence) and by highperformance liquid chromatography; the product ratio of cGDPR/GDP-ribose was 2:1. Bovine spleen NAD ؉ glycohydrolase is also able to hydrolyze cADPR ( Cyclic ADP-ribose, which was originally discovered in sea urchin eggs, is thought to be the endogenous regulator of the Ca 2ϩ -induced Ca 2ϩ