Transient state kinetic evidence for an oligomer in the mechanism of Na+-H+ exchange.

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RESUMO

Pre-steady-state kinetic measurements of 22Na+ uptake by the amiloride-sensitive Na+-H+ exchanger in renal brush border membrane vesicles (BBMV) were performed at 0 degrees C to characterize the intermediate reactions of the exchange cycle. At 1 mM Na+, the initial time course of Na+ uptake was resolved into three separate components: (i) a lag phase, (ii) an exponential or "burst" phase, and (iii) a constant velocity or steady-state phase. Pulse-chase experiments using partially loaded BBMV showed no evidence for 22Na+ back-flux, suggesting that the decline in the rate of Na+ uptake rate following the burst represents completion of the first turnover of the exchanger. Gramicidin completely abolished Na+ uptake, indicating that the burst phase results from the translocation of Na+ rather than from residual Na+ binding to external sites. Raising the [Na+] from 1 to 10 mM at constant pH (internal pH 5.7; external pH 7.7) produced a sigmoidal increase in the amplitude of the burst phase without affecting the lag duration or the apparent burst rate. In contrast, Na+ uptake in the steady state obeyed Michaelis-Menten kinetics. These results suggest that a minimum of two Na+ transport sites must be occupied to activate Na+ uptake in the pre-steady state. The transition to Michaelis-Menten kinetics in the steady state can be explained by a "flip-flop" or alternating site mechanism in which the functional transport unit is an oligomer and only one promoter per cycle is allowed to form a translocation complex with Na+ after the first turnover.

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