Function of NAD glycohydrolase in ADP-ribose uptake from NAD by human erythrocytes

The function of the ectoenzyme NAD glycohydrolase (NADase) in ADP-ribose uptake from extracellular NAD was studied in human erythrocytes that express relatively high NADase activity (adult erythrocytes) and erythrocytes expressing very low activity (newborn erythrocytes). The rates of ADP-ribose uptake from NAD in human erythrocytes were correlated with their NADase activities. In contrast, there was no significant difference in the rates of ADP-ribose uptake among these cells when incubated with ADP-ribose. These results indicate that ecto-NADase may have a role as supplier of ADP-ribose for its uptake into the cells and that the cleavage of NAD by NADase is necessary for the ADP-ribose uptake by human erythrocytes. From ADP-ribose uptake studies at 37δC a K_m of 0.7 ± 0.05 μM and a V_max of 2.04 ± 0.1 pmol/min per μl cell water was found for the uptake of [³H]ADP-ribose. The thiol-reactive reagents p-chloromercuribenzene sulfonic acid and N-ethylmaleimide inhibited the uptake ADP-ribose with IC₅₀ values of 50 ± 4 and 750 ± 25 mM, respectively. Since efflux of [³H]ADP-ribose was negligible, the ADP-ribose transport system appears to be unidirectional. The unidirectionality was supported by the evidence that transported ADP-ribose was rapidly degraded to AMP which is impermeable to the membrane.