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  • A novel method to differentiate between ping-pong and simultaneous exchange kinetics and its application to the anion exchanger of the HL60 cell.

A novel method to differentiate between ping-pong and simultaneous exchange kinetics and its application to the anion exchanger of the HL60 cell.

The Journal of general physiology (1992-11-01)
D Restrepo, B L Cronise, R B Snyder, P A Knauf
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Lithium 3,5-diiodosalicylate, analytical standard
Sigma-Aldrich
3,5-Diiodosalicylic acid, 99%