kinetic analysis of antiport data

david salt BOTSALT at VM.UOGUELPH.CA
Thu Jan 14 14:14:22 EST 1993


 I have been working on a Cd/H antiport at the tonoplast of oat
roots and I have some kinetic data which I am not sure how to
interpret, could anybody help?

I am working with isolated tonoplast vesicles which I load with
H using the V-type ATPase. I measure the initial rate of H
accumulation using absorbance decrease at 490nm of acridine
orange. The outward movement of H from the vesicles is then
coupled to Cd inward movement (measured directly using 109Cd).

The data I am having problems with is the initial rate of H
accumulation at different [ATP] AND different [Cd]. What kinetic
parameters if any can I obtain from this data.

If I plot this data as a v vs S or Eadie Hoftsee I get what
appears to be competative inhibition of H accumulation by Cd
(due to antiport activity, at the [Cd] used Cd has no effect of
ATPase directly), is the Ki for Cd then equivalent to the Km for
Cd transport (Km for Cd transport 5 micromolar measured directly
using 109Cd and Ki for Cd approx. 10 micromolar).

Could I view this as a dual substrate system, with ATP and Cd
being the substrates and H accumulation the product? Is there
some plot which would then allow me to obtain a Km for both ATP
and Cd?

I have not seen anybody try to analyses this type of antiport
data before.



David Salt

Botany Department

University of Guelph

Guelph, Ontario, Canada

Botsalt at vm.uoguelph.ca







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