A pyrophosphatase which degrades NAD+ is located on the external surface of cultured fibroblasts: evidence that NAD+ is not extruded during treatment with N-methyl-N'-nitro-N-nitrosoguanidine.
The Harvey sarcoma virus-transformed derivative of normal rat kidney cells contains a very large membrane-associated NAD+ pyrophosphatase activity. Exogenous NAD+ was metabolized into NMN, demonstrating that the enzyme was located, at least in part, on the external surface of the cell. This external location of the pyrophosphatase was used to evaluate possible extrusion of NAD+ during treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); i.e., extruded NAD+ would be degraded to NMN. NAD+ levels quickly decreased following MNNG addition. The nicotinamide moiety of degraded NAD+ was accounted for as nicotinamide in the culture medium, and no NMN was detectable. Thus, NAD+ was not extruded during MNNG treatment. Additional studies were done to determine which enzyme system was responsible for the rapid fall in NAD+ in these cells. MNNG treatment increased (ADP-rib)n synthetase activity; moreover, this increase in activity was sufficient to account for the loss in intracellular NAD+. Also, the decrease in NAD+ during treatment with MNNG was prevented by 3-aminobenzamide, an agent which inhibited (ADP-rib)n synthetase but not NAD+ glycohydrolase or pyrophosphatase. MNNG had no effect on NAD+ glycohydrolase or pyrophosphatase activities. The results are consistent with the proposal that NAD+ is not extruded but rather is rapidly metabolized by the nuclear (ADP-rib)n synthetase during MNNG treatment.
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D005347:Fibroblasts / Q000201:enzymology; D007668:Kidney; D007700:Kinetics; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009244:NAD+ Nucleosidase; D011755:Pyrophosphatases / Q000378:metabolism*; D051381:Rats
D008769:Methylnitronitrosoguanidine; D009699:N-Glycosyl Hydrolases; D009244:NAD+ Nucleosidase; D011755:Pyrophosphatases; C024289:NAD pyrophosphatase