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

• DOI: 10.1073/pnas.86.13.4818
• Publication Date: 2006-05-31T10:58:01Z
• Authors: K Otsu; J Kinsella; B Sacktor; J P Froehlich
• Keywords: Amiloride

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