Dan Cassel1, Thomas Pfeuffer1
1Department of Physiological Chemistry, University of Würzburg, Medical School, 8700 Würzburg, Federal Republic of Germany
Tóm tắt
Treatment of pigeon erythrocyte membranes with cholera toxin and NAD
+
enhanced the GTP stimulation and suppressed the F
-
activation of the adenylate cylase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. In the presence of NAD
+
labeled with
32
P in the AMP moiety the toxin catalyzed the covalent incorporation of radioactivity into membrane proteins with molecular weights (
M
r
s) of 200,000, 86,000, and 42,000. Extraction of toxin-treated membranes with Lubrol PX followed by affinity chromatography on a GTP-Sepharose column resulted in a 200-fold purification of the 42,000-
M
r
labeled protein and in its complete separation from the other labeled proteins. The fraction containing the purified GTP-binding component from toxin-treated membranes conferred an enhanced GTP-stimulated activity on adenylate cyclase solubilized from nontreated membranes. Likewise, the addition of GTP-binding fraction from nontreated membranes to an enzyme solubilized from toxin-treated membranes restored F
-
stimulation of the adenylate cyclase. The toxin-induced modification of adenylate cyclase and the incorporation of radioactivity into the 42,000-
M
r
protein were partially reversed upon incubation with toxin and nicotinamide at pH 6.1. The results indicate that cholera toxin affects the adenylate cyclase system by catalyzing an ADP-ribosylation of the 42,000-
M
r
component bearing the guanyl nucleotide regulatory site.
