An 18-kDa domain of a glycosylphosphatidylinositol-linked NAD:arginine ADP-ribosyltransferase possesses NAD glycohydrolase activity.

Abstract

Transfection of NMU (rat mammary adenocarcinoma) cells with NAD:arginine ADP-ribosyltransferase cDNAs from Yac-1 murine lymphoma cells or rabbit muscle increased NAD glycohydrolase and ADP-ribosyltransferase activities. The ADP-ribosyltransferase activity was released from transformed NMU cells by phosphatidylinositol-specific phospholipase C (PI-PLC) 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 transferase was approximately 40 kDa and that of the detergent-solubilized NADase was approximately 100 kDa. Using polyclonal antibodies against rabbit muscle transferase on Western blots, approximately 18- and approximately 30-kDa band were visualized among proteins from the NADase fractions and 38-40-kDa bands with protein from the transferase fractions. Incubation of blots with [32P]NAD led to the incorporation of radioactivity into the immunoreactive transferase bands of 38 kDa and the immunoreactive NADase band of approximately 18 kDa. These data suggest that proteolysis of ADP-ribosyltransferase synthesized in transformed NMU cells might result in the formation of aggregates of an 18-kDa NAD glycohydrolase. A fusion protein with glutathione S-transferase linked to the amino terminus of Yac-1 transferase, from which the amino-terminal 121 amino acids had been deleted (GST-Yac-1-delta121), 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 transformed with transferase cDNA. These results are compatible with the conclusion that the NAD glycohydrolase activity was generated in NMU cells by proteolysis of ADP-ribosyltransferase, with release of a carboxyl-terminal fragment that possesses glycohydrolase but not transferase activity, i.e. the carboxyl-terminal portion of the transferase can exist as a catalytically active NADase.

• Authors: H J Kim, I J Okazaki, T Takada, J Moss
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 272 Issue 14 Pg. 8918-23 (Apr 04 1997) ISSN: 0021-9258 [Print] United States
• PMID: 9083012 (Publication Type: Journal Article)

Chemical References

• DNA, Complementary
• Glycosylphosphatidylinositols
• ADP Ribose Transferases
• Poly(ADP-ribose) Polymerases
• NAD+ Nucleosidase

Topics

• ADP Ribose Transferases (chemistry, metabolism)
• Adenocarcinoma (enzymology)
• Animals
• Chromatography, Gel
• Chromosomes, Artificial, Yeast
• DNA, Complementary (metabolism)
• Female
• Glycosylphosphatidylinositols (metabolism)
• Mammary Neoplasms, Experimental (enzymology)
• Molecular Weight
• Muscle, Skeletal (enzymology)
• NAD+ Nucleosidase (metabolism)
• Poly(ADP-ribose) Polymerases (genetics, metabolism)
• Rabbits
• Rats
• Transfection
• Tumor Cells, Cultured