Isolation of an avian erythrocyte protein possessing ADP-ribosyltransferase activity and capable of activating adenylate cyclase

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Abstract
An ADP-ribosyltransferase was purified .apprx.500-fold from the supernatant fraction of turkey erythrocytes. The enzyme hydrolyzed [carbonyl-14C]NAD to ADP-ribose and [carbonyl-14C]nicotinamide at a low rate. Nicotinamide formation from NAD was enhanced by arginine methyl ester > D-arginine .apprx. L-arginine > guanidine; lysine, histidine and citrulline were ineffective. Incubation of [adenine-U-14C]NAD and arginine methyl ester or arginine with the purified enzyme resulted in the formation of new compounds that contained 14C, reacted with ninhydrin and quenched background fluorescence of thin-layer plates viewed in UV light. Their mobilities on TLC were indistinguishable from those of ADP-ribosylarginine methyl ester and ADP-ribosylarginine formed during incubation of choleragen with NAD and arginine methyl ester or arginine, respectively. The purified transferase also catalyzed the incorporation of label from [adenine-14C]-NAD into lysozyme, histones and polyarginine. When the 14C-labeled lysozyme was incubated with snake venom phosphodiesterase, the radioactivity was released and, on TLC, exhibited a mobility indistinguishable from that of 5''-AMP, as would be expected of an ADP-ribosylated protein, but not of a poly(ADP-ribosylated) product. The purified transferase activated rat brain adenylate cyclase and, as is the case with choleragen, activation was absolutely dependent on NAD. The presence in the avian erythrocyte of a protein that, like choleragen and Escherichia coli heat-labile enterotoxin, apparently activates adenylate cyclase and possesses ADP-ribosyl transferase activity is consistent with the view that the mechanisms through which the bacterial toxins produce pathology are not entirely foreign to vertebrate cells, at least some of which may possess and employ an analogous mechanism for activation of adenylate cyclase.

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