kinetic analysis of antiport data
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
I have not seen anybody try to analyses this type of antiport
University of Guelph
Guelph, Ontario, Canada
Botsalt at vm.uoguelph.ca
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