Improvement of cholera toxin-catalyzed ADP-ribosylation by endogenous ADP-ribosylation factor from bovine brain provides evidence for an unchanged amount of Gs alpha in failing human myocardium.
The aim of the present study was to investigate whether or not alterations of Gs alpha can be detected with cholera toxin-induced ADP-ribosylation in myocardial membranes from patients with heart failure. Therefore, Gs alpha was radiolabeled by cholera toxin-catalzyed (32P)ADP-ribosylation with (32P)NAD as substrate. In membranes from left ventricular myocardium of six patients with dilated cardiomyopathy classified as NYHA IV and three samples from two non-failing donor hearts, labeling was too weak to allow detection of possible changes in the amount of Gs alpha. Therefore, the cytosolic small molecular weight G protein ARF (ADP-ribosylation factor), a cofactor for cholera toxin-induced ADP-ribosylation of Gs alpha, was partially purified from bovine cerebral cortex. ARF activity was quantified by its ability to enhance auto-ADP-ribosylation of cholera toxin A1-subunit. Gs alpha was identified by comparing the ADP-ribosylation patterns of myocardial membranes, membranes prepared from human leukemia (HL 60) and S 49 mouse lymphoma wild type cells (45 kDa-band present) with membranes of the Gs alpha-deficient S 49 variant cyc- (45 kDa-band missing). In the presence of ARF, specific radiolabeling of the Mr 45,000 subtype of Gs alpha was markedly enhanced. The amounts of Gs alpha as measured by cholera toxin-dependent (32P)-ADP-ribosylation in the presence of ARR were similar in failing and nonfailing human hearts. It is concluded that factors other than Gs alpha are responsible for the altered regulation of the adenylate cyclase complex in heart failure. Moreover, by enhancing cholera toxin-catalyzed ADP-ribosylation, endogenous ADP-ribosylation factor from bovine brain appears to be a useful tool to study Gs alpha even in tissues in which the labeling of Gs alpha is rather weak.
Schnabel
P
P
Medizinische Klinik I, Universität München, FRG.
Böhm
M
M
Gierschik
P
P
Jakobs
K H
KH
Erdmann
E
E
D020727:ADP-Ribosylation Factors; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002540:Cerebral Cortex / Q000032:analysis; D002772:Cholera Toxin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006333:Heart Failure / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism
D008565:Membrane Proteins; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors