The 65-kDa protein from pig heart. A new substrate for Clostridium botulinum ADP-ribosyltransferase (exoenzyme C3).

In the pig heart sarcolemma, a 65 kDa protein is found to be ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase (exoenzyme C3). ADP-ribosylation of this protein is regulated by guanyl nucleotides and cytosol factor in a fashion similar to that for other C3 substrates. The new exoenzyme C3 substrate was partially purified. This protein is supposed to be a GTP-binding one.

Dicyclohexylcarbodiimide influences the regulatory properties of the adenylate cyclase system in rat reticulocyte membranes.

Treatment of rat reticulocyte plasma membranes with dicyclohexylcarbodiimide (DCCD) decreased the GTP-stimulated adenylate cyclase activity and reduced the number of receptors that bind the agonist with high affinity in the absence of GTP. Besides, the agonist's competition curve was shifted to the right, irrespective of the presence of guanyl nucleotides. The dissociation constant for the antagonist and the number of binding sites did not change. Preincubation of the DCCD-treated membranes with GMP in the presence of isoproterenol restored the regulation of the agonist's affinity by guanyl nucleotides; however, in this case the GTP-independent change in the agonist's affinity was retained. This suggests that one of the DCCD-modified components of the adenylate cyclase system is a regulatory protein.

Purification and some properties of GTP-binding proteins from pig heart plasma membranes.

The major GTP-binding proteins (N-proteins) have been purified from cholate extract of pig heart plasma membranes using chromatography through DEAE-Trisacryl, Sephacryl S-300, Octyl-Sepharose, and DEAE-Sepharose. The hydrophobic chromatography resulted in elution of two GTP-binding activity peaks. The specific GTP gamma S binding of both N-protein preparations was 2.5 nmol/mg of protein with KD value 2.10(-7) M. The first preparation contained three polypeptides with molecular masses 40 kDa, 36 kDa, and 23 kDa. 40 kDa polypeptide was ADP-ribosylated by pertussis toxin. The same protein appeared to be the only substrate of pertussis toxin in crude detergent extract of membranes. The major polypeptides of the second preparation were represented by 37 kDa and 23 kDa polypeptides.

Signal-transducing GTP-binding proteins of mammalian heart and lungs.

Signal-transducing GTP-binding Proteins of Mammalian Heart and Lungs. Journal of Molecular and Cellular Cardiology (1989) 21 (Suppl I) 91-95. Three distinct G-proteins have been found in mammalian heart sarcolemma: Gi (alpha i = 40 kDa, beta = 36 kDa, and lambda less than 14 kDa), Gp (alpha p = 23 kDa, beta = 36 kDa, and lambda less than 14 kDa), and Gs (alpha s = 42 kDa). ADP-ribosylation of sarcolemmal alpha i by pertussis toxin (PT) or preincubation of sarcolemma with protein kinase C and PMA resulted in increased adenylate cyclase activity and blockade of GTP-dependent inhibition by carbachol whereas the GTP-dependent activating effect of isoproterenol on the adenylate cyclase was preserved. ADP-ribosylation of alpha i in sarcolemma by endogenous NADP-sensitive ADP-ribosyltransferase abolished the PT-induced ADP-ribosylation of alpha i. Gpp (NH)p attenuated the PT-induced ADP-ribosylation of alpha i and promoted the cholera toxin (CT)-induced ADP-ribosylation of alpha s. The CT-induced alpha s ADP-ribosylation was enhanced in the presence of heart cytosol. Soluble Gi- and Gs-proteins were identified in lung cytosol. The 40 kDa alpha i in membrane and soluble fractions was ADP-ribosylated by PT, while the soluble 42 kDa alpha s was ADP-ribosylated by CT in lung tissue. The ADP-ribosylation of soluble alpha i by PT-suppressed guanyl nucleotide binding to Gi. The apparent molecular mass of partially purified soluble Gi was 75 kDa.