PLANT PHYSIOLOGY , Vol 101, Issue 1 127-133, Copyright © 1993 by American Society of Plant Biologists
Inhibition of Thylakoid ATPase by Venturicidin as an Indicator of CF1-CF0 Interaction
S. Zhang, D. D. Letham and A. T. Jagendorf
Section of Plant Biology, Cornell University, Ithaca, New York 14850
Venturicidin inhibits the F0 portion of membrane-located, H+-pumping
ATPases. We find it meets the criteria for an energy transfer inhibitor for
spinach (Spinacia oleracea) thylakoids: complete inhibition of
photophosphorylation and of photophosphorylation-stimulated and basal
electron flow rates, but not of electron flow under uncoupled conditions.
The extent of H+ uptake in the light is stimulated by venturicidin (vtcd),
as expected for a compound blocking H+ efflux through CF0. Vtcd had no
effect on the nonproton pumping, methanol-stimulated ATPase of thylakoids
or on soluble CF1 ATPase. Under totally uncoupled conditions (saturating
NH4Cl + gramicidin), vtcd can still inhibit sulfite-stimulated thylakoid
ATPase completely. The concentration of vtcd needed for inhibition of
ATPase was proportional to the concentration of thylakoids present in the
assay, with an apparent stoichiometry of about 10 vtcd molecules per
CF1/CF0 for 50% inhibition. Vtcd raised the Km for ATP somewhat, but had a
stronger effect on the Vmax with respect to ATP. Inhibition by saturating
vtcd ranged from 50 to 100%, depending on the condition of the thylakoids.
Grinding leaves in buffer containing 0.2 M choline chloride (known to
provide superior photophosphorylation rates) helped bring on maximum vtcd
inhibition; trypsin treatment or aging of thylakoids brought on
vtcd-resistant ATPase. We conclude that the extent of inhibition by vtcd
can be used as an indicator of the tightness of coupling between CF1 and
CF0.
