The effects of hydrostatic pressure on the low-K m GTPase in brain membranes from two congeneric marine fishes

To investigate, the effects of hydrostatic pressure on transmembrane signaling in cold-adapted marine fishes, we examined the high-affinity GTPase activity in two congeneric marine fishes, Sebastolobus alascanus and S. altivelis. In brain membranes there are two GTPase activities, one with a low K _m and one with a high K _m for GTP. The high-affinity GTPase activity, characteristic of the subunits of the guanine nucleotide binding protein pool, was stimulated by the A₁ adenosine receptor agonists N ⁶(R-phenylisopropyl)adenosine and N ⁶-cyclopentyladenosine, and the muscarinic cholinergic agonist carbamyl choline. Pertussis toxin-catalyzed ADP-ribosylation of the membranes for 2 h at 5°C prior to the GTPase assay decreased the basal GTPase activity 30–40% and abolished N ⁶ (R-phenylisopropyl)adenosine stimulation of GTP hydrolysis. Basal high-affinity hydrolysis of GTP, measured at 0.3 mol·1^-1GTP, was stimulated 22% in both species by 340 atm pressure. At 340 atm pressure, the apparent K _m of GTP is decreased approximately 10% in each of the species, and the V _max values are increased 11 and 15.9% in S. alascanus and S. altivelis, respectively. The apparent volume changes associated with the decreased K _m of GTP and the increased V _max ranged from-7.0 to-9.9 ml·mol^-1. Increased pressure markedly decreased the efficacy of N ⁶ (R-phenylisopropyl) adenosine, N ⁶-cylcopentyladenosine and carbamyl choline in stimulating GTPase activity. The effects of increased hydrostatic pressure on transmembrane signal transduction by the A₁ adenosine receptor-inhibitory guanine nucleotide binding protein-adenylyl cyclase system may stem, at least in part, from pressure-increased GTP hydrolysis and the concomitant termination of inhibitory signal transduction.Abbreviations ³H] DPCPX ³H cyclopentyl-1, 3-dipropylxanthine - AppNHp 5-adenylylimidodiphosphate - cpm counts per minute - CPA N ⁶-cyclopentyladenosine - EDTA ethylenediaminetetra acetic acid - EGTA ethyleneglycol-bis (-aminoethylether) N, N, N, N-totra-acctic acid - G protein guanine nucleotide binding protein - G_i inhibitory G protein - G_o other G protein, common in brain membranes - G_s stimulatory G protein - GTPase guanosine triphosphatase - K _i inhibition constant - K _m Michaelis constant - pK _a log of the dissociation constant - R-PIA N ⁶ (R-phenylisopropyl) adenosine - TRIS trishydroxymethyl]aminomethane - V_max maximal velocity - -³²P]GTP -³²P] guanosine 5-triphosphate (tetra (triethylammonium) salt)