Abstract
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.
Full Text
The Full Text of this article is available as a PDF (690.0 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bakker-Grunwald T., van Dam K. The energy level associated with the light-triggered Mg 2+ -dependent ATPase in spinach chloroplasts. Biochim Biophys Acta. 1973 Apr 5;292(3):808–814. doi: 10.1016/0005-2728(73)90027-3. [DOI] [PubMed] [Google Scholar]
- Binder A., Jagendorf A., Ngo E. Isolation and composition of the subunits of spinach chloroplast coupling factor protein. J Biol Chem. 1978 May 10;253(9):3094–3100. [PubMed] [Google Scholar]
- Gould J. M. Inhibition by triphenyltin chloride of a tightly-bound membrane component involved in photophosphorylation. Eur J Biochem. 1976 Mar 1;62(3):567–575. doi: 10.1111/j.1432-1033.1976.tb10191.x. [DOI] [PubMed] [Google Scholar]
- Kovác L., Hrusovská E. Oxidative phosphorylation in yeast. II. An oxidative phosphorylation-deficient mutant. Biochim Biophys Acta. 1968 Jan 15;153(1):43–54. doi: 10.1016/0005-2728(68)90144-8. [DOI] [PubMed] [Google Scholar]
- LeBel D., Poirier G. G., Beaudoin A. R. A convenient method for the ATPase assay. Anal Biochem. 1978 Mar;85(1):86–89. doi: 10.1016/0003-2697(78)90277-4. [DOI] [PubMed] [Google Scholar]
- Lill H., Junge W. CF0, the proton channel of chloroplast ATP synthase. After removal of CF1 it appears in two forms with highly different proton conductance. Eur J Biochem. 1989 Feb 1;179(2):459–467. doi: 10.1111/j.1432-1033.1989.tb14575.x. [DOI] [PubMed] [Google Scholar]
- McCarty R. E., Guillory R. J., Racker E. Dio-9, an inhibitor of coupled electron transport and phosphorylation in chloroplasts. J Biol Chem. 1965 Dec;240(12):4822–4823. [PubMed] [Google Scholar]
- Nelson N. Structure and function of chloroplast ATPase. Biochim Biophys Acta. 1976 Nov 30;456(3-4):314–338. doi: 10.1016/0304-4173(76)90003-3. [DOI] [PubMed] [Google Scholar]
- Pick U., Racker E. Purification and reconstitution of the N,N'-dicyclohexylcarbodiimide-sensitive ATPase complex from spinach chloroplasts. J Biol Chem. 1979 Apr 25;254(8):2793–2799. [PubMed] [Google Scholar]
- Sigrist-Nelson K., Sigrist H., Azzi A. Characterization of the dicyclohexylcarbodiimide-binding protein isolated from chloroplast membranes. Eur J Biochem. 1978 Dec 1;92(1):9–14. doi: 10.1111/j.1432-1033.1978.tb12717.x. [DOI] [PubMed] [Google Scholar]
- Wintermans J. F., de Mots A. Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol. Biochim Biophys Acta. 1965 Nov 29;109(2):448–453. doi: 10.1016/0926-6585(65)90170-6. [DOI] [PubMed] [Google Scholar]