AJP - Cell Physiology, Vol 259, Issue 3 C490-C502, Copyright © 1990 by American Physiological Society
Essential activation of Na(+)-H+ exchange by [H+]i in HL-60 cells
D. Restrepo, D. S. Cho and M. J. Kron
Monell Chemical Senses Center, Philadelphia, Pennsylvania 19104.
The intracellular pH (pHi) dependence of the rate of Na(+)-H+ exchange was
determined in undifferentiated promyelocytic HL-60 cells by measuring
alkalinization rates using the fluorescent pHi indicator
2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). BCECF was
calibrated in the pH range from 5 to 7 using the nigericin technique of
Thomas and co-workers (J. A. Thomas, R. N. Buchsbaum, A. Zimniak, and E.
Racker. Biochemistry 18: 2210-2218, 1979). Exchange rate increases as pHi
is lowered below pH 7.00. At low pH (pH below 6.3), the dependence of
Na(+)-H+ exchange rate on intracellular proton activity is well fitted by
the Michaelis-Menten equation with a maximum exchange velocity of 33.7 +/-
2.4 mmol H(+).1 cell water-1.min-1 and a half-saturation constant of 1.35
+/- 0.28 microM (corresponding to a minus log of the Michaelis constant of
5.89). However, a Hill plot reveals that the Hill coefficient changes
gradually from one to two when pH is changed from 5 to 7, ruling out
Michaelian kinetics. The dependence of exchange flux on internal protons is
well fit in the full pH range from 5 to 7 by a simple kinetic model
(essential activation) with modifier and transport sites for internal
proton binding. At low pH, failure to correct BCECF measurement of pHi for
contribution to fluorescence signal from extracellular dye and for
quenching of intracellular BCECF leads to an artifactual increase in the
measured Hill coefficient. These two findings (increase in Hill coefficient
as pHi is increased and artifactual increase in Hill coefficient because of
methodological reasons) provide a good explanation for the wide range of
Hill coefficients reported in the literature.
