Me. Cavet et al., Na+/H+ exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface, AM J P-CELL, 277(6), 1999, pp. C1111-C1121
NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian
Na+/H+ exchanger (NHE) gene family. Given the specialized function and reg
ulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1
, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lackin
g endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular
stomatitis virus glycoprotein (VSVG). The following characteristics were de
termined on the same passage of cells transfected with NHE1V, NHE2V, or NHE
3V: 1) maximal reaction velocity (V-max) by Na-22(+) uptake and fluorometer
y, 2) total amount of NHE protein by quantitative Western analysis with int
ernal standards of VSVG-tagged maltose-binding protein, and 3) cell surface
expression by cell surface biotinylation. Cell surface expression (percent
age of total NHE) was 88.8 +/- 3.5, 64.6 +/- 3.3, 20.0 +/- 2.6, and 14.0 +/
- 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite the
se divergent cell surface expression levels, turnover numbers for NHE1, NHE
2, and NHE3 were similar (80.3 +/- 9.6, 92.1 +/- 8.6, and 99.2 +/- 9.1 s(-1
), when V-max was determined using Na-22 uptake at 22 degrees C and 742 +/-
47, 459 +/- 16, and 609 +/- 39 s(-1) when V-max was determined using fluor
ometry at 37 degrees C). These data indicate that, in the same cell system,
intrinsic properties that determine turnover number are conserved among NH
E1, NHE2, and NHE3.