INVESTIGADORES
GONZALEZ LEBRERO Rodolfo Martin
congresos y reuniones científicas
Título:
Looking for effects of [Rb+] on the rate deocclusion from the Na,K-ATPase
Autor/es:
SB. KAUFMAN; RM. GONZÁLEZ LEBRERO; RC. ROSSI; JENS G, NOERBY; PJ. GARRAHAN
Lugar:
Buenos Aires, Argentina,
Reunión:
Congreso; IIIrd Iberoamerican congress of Biophyics; 1997
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
It is currently accepted that active transport of K+ by the Na,K-ATPase occurs through the binding of two extracellular K+ to a phosphorylated intermediate of the pump (E2P), followed by dephosphorylation and by the simultaneous occlusion of K+ with formation of E2(K2). Release of K+ from E2(K2) is slow, but the binding of ATP to E2(K2) accelerates the reaction more than 200 times. The Km for the nucleotide on this effect is 300-500 µM. In this scheme, the ratio ?activity/(E2(K2) + E2(K2)ATP)? (i.e. v/Eocc) in steady state equals the rate coefficient for deocclusion (kdeocc), provided that hydrolysis of ATP occurs only through the formation and breakdown of occluded intermediates and deocclusion is irreversible. We measured the ratio v/Eocc using the potassium congener Rb+ (as 86Rb) at 0.5 mM ATP and concentrations of Rb+ from 0.25 to 10 mM, and compared the values with a direct measurement of kdeocc from the time course of 86Rb deocclusion. Occluded rubidium was measured as described by Rossi and Nørby (J. Biol. Chem., 282: 12579-12590). Determination of kdeocc was performed by isotopic dilution of a medium with (86Rb)-occluded enzyme (at least 20 fold), either keeping constant [Rb+] (which required to dilute it in 20 volumes of the same medium, but lacking 86Rb), or by increasing the concentration of (unlabelled) Rb+. Experiments were performed at 25C using Na,K-ATPase purified from pig kidney, in media containing 150 mM NaCl, EDTA 200 µM, enough MgCl2 to keep [Mg2+] free at 0.5 mM and Imidazol-HCl 25 mM, pH = 7.4. Results showed that the ratio v/Eocc decreased asymptotically from 48  4.7 s-1 to 30  0.17 s-1 as [Rb+] increased from 0.25 to 10 mM, while kdeocc was 18.0  1.5 s-1, regardless of the concentration of Rb+. Further, kdeocc remained constant, irrespectively on whether the concentration of rubidium was kept constant or not after isotopic dilution. This results contrast with previous findings by Forbush (J. Biol. Chem., 262: 11104-11115), who reported that, at milimolar concentrations of ATP, K+ was able to increase the rate of deocclusion of 86Rb+ up to 60%.With grants from the European Union and Universidad de Buenos Aires.