A novel method to differentiate between ping-pong and simultaneous exchange kinetics and its application to the anion exchanger of the HL60 cell.
We have developed a new test to differentiate between ping-pong and simultaneous mechanisms for tightly coupled anion exchange. This test requires the use of a dead-end reversible noncompetitive inhibitor. As an example, we have applied the test to the anion exchanger of the HL60 cell using the salicylic acid derivative 3,5-diiodosalicylic acid (DIS), which reversibly inhibits HL60 cell Cl/Cl exchange. The concentration of DIS that causes 50% inhibition (ID50) increased only slightly as either intra- or extracellular chloride was increased, indicating that DIS inhibits HL60 anion exchange in a noncompetitive manner. In agreement with this observation, plots of the slope of the Dixon plot as a function of 1/[Clo] or 1/[Cli] were fit with straight lines with nonzero intercepts, indicating that DIS does not compete with either of the substrates ([Clo] and [Cli]). The secondary Dixon slope test is based on the fact that, for a dead-end inhibitor such as DIS, the slope of the Dixon plot slope vs. 1/[Cli] (secondary Dixon slope or SDS) is independent of extracellular Cl when the exchange mechanism follows ping-pong kinetics. Similarly, the SDS calculated from a plot as a function of 1/[Clo] is also independent of intracellular Cl for a ping-pong exchanger. In contrast to this prediction, we found that for DIS inhibition of Cl/Cl exchange in HL60 cells the slope of the Dixon plot slope vs. 1/[Cli] decreased by a factor of 2.5-fold when [Clo] was increased from 1 to 11 mM (P < 0.0001). This change in the SDS rules out ping-pong kinetics, but is consistent with a simultaneous model of Cl/Cl exchange in which there are extra- and intracellular anion binding sites, both of which must be occupied by suitable anions in order to allow simultaneous exchange of the ions.
Restrepo
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Monell Chemical Senses Center, Philadelphia, Pennsylvania 19104.
Cronise
B L
BL
Snyder
R B
RB
Knauf
P A
PA
D001639:Bicarbonates / Q000378:metabolism; D001665:Binding Sites; D002712:Chlorides / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007463:Iodobenzoates; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D008954:Models, Biological; D012459:Salicylates / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*
D001639:Bicarbonates; D002712:Chlorides; D007463:Iodobenzoates; D012459:Salicylates; C020891:3,5-diiodosalicylic acid