The effects of hydrostatic pressure on pertussis toxin-catalyzed ribosylation of G proteins from deep-living macrourid fishes

To test the effects of hydrostatic pressure on the coupling of receptors to guanyl nucleotide binding reglatory proteins (G proteins) in transmembrane signaling, pertussis toxin (PTX)-catalyzed [P-32]ADP-ribosylation was used to probe the guanyl nucleotide-binding proteins G(i) and G(o) in brain mem branes from four marine teleosts. These macrourids, Coryphaenoides pectoral is, Coryphaenoides cinereus, Coryphaenoides filifer and Coryphaenoides arma tus, span depths from 200 to 5400 m. Pertussis toxin specifically labelled proteins of 39-41 kDa. The PTX-catalyzed [P-32]ADP-ribosylation reaction wa s linear for 7 h. Added guanyl nucleotides (guanosine 5'-diphosphate (GDP) and guanosine 5'-O-(3-thio-tri phosphate)(GTP[S])) at concentrations up to 1000 mu M did not affect ribosylation at atmospheric pressure. Under basal conditions the G(i)/G(o) protein population appears to be uncoupled from re ceptors and bound with GDP. Pressures up to 476 atm were tested in the abse nce and presence of added guanyl nucleotides, 100 mu M GDP and 100 mu M GTP [S]. [P-32]ADP-ribosylation in brain membranes from the deeper-occurring C. cinereus, C. filifer and C. armatus was not inhibited by increased pressur e in the presence of 100 CIM GDP. Increasing pressure decreased ribosylatio n in brain membranes of C. pectoralis. In the presence of 100 mu M GTP[S], increased pressure inhibited ribosylation in all species. Pressure appears to enhance the efficacy of GTP[S] in dissociating the heterotrimeric holopr otein. (C) 2000 Elsevier Science Inc. All rights reserved.