NAD glycohydrolase and ADP-ribosyltransferase activities are intrinsic to the A 1 peptide of choleragen

Abstract

In prior reports (Moss, J., Manganiello, V. C., and Vaughan, M. (1976) Proc. Natl. Acd Sci. U. S. A. 73, 4424-4427), it was demonstrated that choleragen preparations possessed NAD glycohydrolase activity. Tait and Van Heyningen ((1978) Biochem. J. 174,1059-1062) have recently proposed that the activity observed in these preparations was due to a contaminating enzyme and was not an intrinsic activity of choleragen. The absence of an NAD glycohydrolase activity in choleragen preparation would imply that nicotinamide release from NM requires a ternary complex of NAD, acceptor protein, and toxin and that, in contrast to diphtheria toxin and Pseudomonas exotoxin A, choleragen cannot use water as an ADP-ribose acceptor. Since the ability to hydrolyze NAD is critical to understanding the mechanism of the toxin-catalyzed reaction, we have re-examined the question using purified choleragen peptides. In agreement with our prior observations, the purified A1 peptide of choleragen, but not the AZ or B, catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide and the NAD-dependent ADP-ribosylation of arginine. The NAD glycohydrolase activity cochromatographed with the A1 peptide, the ADP-ribosyltransferase activity, and the ability to activate rat liver adenylate cyclase. The K, for NAD was similar for the holotoxin and A1. The turnover number of A1 was similar to that of the intact toxin which contains one A1 per molecule. In contrast to the holotoxin or its A subunit, the A1 peptide did not exhibit a lag in reaction rate or a thiol requirement for activity. The enzymatic activity of the A1 peptide was inhibited by sodium dodecyl sulfate as was its ability to activate adenylate cyclase. These results support the conclusion that the A1 peptide of choleragen possesses NAD glycohydrolase activity and can activate the ribosyl-nicotinamide bond of NAD in the absence of an acceptor protein.