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Improvement of cholera toxin-catalyzed ADP-ribosylation by endogenous ADP-ribosylation factor from bovine brain provides evidence for an unchanged amount of Gsα in failing human myocardium

1990; Elsevier BV; Volume: 22; Issue: 1 Linguagem: Inglês

10.1016/0022-2828(90)90973-6

1095-8584

Petra Schnabel,

Calcium signaling and nucleotide metabolism

The aim of the present study was to investigate whether or not alterations of Gsα can be detected with cholera toxin-induced ADP-ribosylation in myocardial membranes from patients with heart failure. Therefore, Gsα was radiolabeled by cholera toxin-catalyzed (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 Gsα. Therefore, the cytosolic small molecular weight G protein ARF (ADP-ribosylation factor), a cofactor for cholera toxin-induced ADP-ribosylation of Gsα, was partially purified from bovine cerebral cortex. ARF activity was quantified by its ability to enhance auto-ADP-ribosylation of cholera toxin A1-subunit. Gsα 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 Gsα-deficient S 49 variant cyc− (45 kDa-band missing). In the presence of ARF, specific radiolabeling of the Mr 45 000 subtype of Gsα was markedly enhanced. The amounts of Gsα as measured by cholera toxin-dependent (32P)-ADP-ribosylation in the presence of ARF were similar in failing and nonfailing human hearts. It is concluded that factors other than Gsα 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 Gsα even in tissues in which the labeling of Gsα is rather weak.