Effect of regulatory anions on the thermal stability of the soluble mitochondrial ATPase (F1)

GASTALDI, LAURA M; LODEYRO, ANABELLA F.; ROVERI, OSCAR A

Rosario, ARGENTINA.

Congreso; XXIII Reunión Anual de la Sociedad de Biología de Rosario; 2003

Sociedad de Biología de Rosario

Mitochondrial ATP synthase catalyzes ATP synthesis driven by an electrochemical gradient generated by the respiratory chain through the inner mitochondrial membrane. It is composed of two sectors: Fo (an oligomer which is integral to the membrane) and F1 ( hydrosoluble oligomer). When F1 is isolated from de membrane is only capable to hydrolize ATP and became cold-labile. F1 is rapidly inactivated when it is kept at 0 ºC, whereas it is stable at 30 ºC; besides, there is no information in the literature of its stability at intermediates temperatures. On the other hand, in our laboratory has been studied the effect of regulatory anions on the ATP-hydrolysis reaction catalized by F1. Bicarbonate behave as a non essential activator while sulfate do as a partial inhibitor at [ATP] < 2 mM  and as a non essential activator at higher concentrations . A reasonable hypothesis for these observations was to postulate that sulfate is binding a catalytic site whereas bicarbonate is binding a non catalytic one. The objective of the present work was to characterize kinetically the thermal stability of F1 and the effect of bicarbonate and sulfate on this. Due to that, it was determinated  the ATPasic activity versus incubation time of the enzyme at temperatures between 0 and 50 ºC, without the presence of regulatory anions and with the presence of sulfate or bicarbonate. At the working incubation temperatures, was observed a single exponential decay with a residual activity that not converge to cero. A qualitatively similar behavior was observed in the presence of bicarbonate or sulfate. However, sulfate produced both, longer half-lives  and  an increase in the residual activities observed, therefore acting as a protecting ligand. Instead, bicarbonate caused a faster inactivation of the enzyme. This result suggest that the conformation of the enzyme with bicarbonate bound is more sensitive both high and low temperatures. Similar results were obtained with three different preparations of the enzyme. Since both anions affect  in different ways the stability of the enzyme,  is possible to conclude that sulfate and bicarbonate bind distinct sites in it. This observation is an experimental evidence that agree with the hypothesis above mentioned: i) sulfate bind a catalytic site instead of phosphate; and  ii) bicarbonate bind a non catalytic site of binding of nucleotides instead of ADP.