AN 18-KDA DOMAIN OF A GLYCOSYLPHOSPHATIDYLINOSITOL-LINKED NAD-ARGININE ADP-RIBOSYLTRANSFERASE POSSESSES NAD GLYCOHYDROLASE ACTIVITY

Transfection of NMU (rat mammary adenocarcinoma) cells with NAD:argini ne ADP-ribosyltransferase cDNAs from Yac-1 murine lymphoma cells or ra bbit muscle increased NAD glycohydrolase and ADP-ribosyltransferase ac tivities. The ADP-ribosyltransferase activity was released from transf ormed NMU cells by phosphatidylinositol-specific phospholipase C (PI-P LC) and hence glycosylphosphatidylinositol (GPI)-anchored, whereas the NAD glycohydrolase (NADase) activity remained cell-associated. By gel permeation chromatography, the size of the PI-PLC-released transferas e was similar to 40 kDa and that of the detergent-solubilized NADase w as similar to 100 kDa. Using polyclonal antibodies against rabbit musc le transferase on Western blots, similar to 18- and similar to 30-kDa band were visualized among proteins from the NADase fractions and 38-4 0-kDa bands with protein from the transferase fractions. Incubation of blots with [P-32]NAD led to the incorporation of radioactivity into t he immunoreactive transferase bands of 38 kDa and the immunoreactive N ADase band of similar to 18 kDa. These data suggest that proteolysis o f ADP-ribosyltransferase synthesized in transformed NMU cells might re sult in the formation of aggregates of an 18-kDa NAD glycohydrolase. A fusion protein with glutathione S-transferase linked to the amino ter minus of Yac-1 transferase, from which the amino-terminal 121 amino ac ids had been deleted (GST-Yac-1-delta 121), exhibited NADase, but not transferase, activity. The size of the recombinant fusion protein was similar to that of the proteolytic fragment seen in NMU cells transfor med with transferase cDNA. These results are compatible with the concl usion that the NAD glycohydrolase activity was generated in NMU cells by proteolysis of ADP-ribosyltransferase, with release of a carboxyl-t erminal fragment that possesses glycohydrolase but not transferase act ivity, i.e. the carboxyl-terminal portion of the transferase can exist as a catalytically active NADase.