ABSTRACT

Under unisite conditions (ratio of ATP to chloroplast coupling factor (CFCF), approximately 1:2.8), spinach thylakoid ATPase depends on prior reductive activation of CF, just as multisite ATPase does, and is sensitive to removal of CF by EDTA. Faster rates in room light than in semidarkness and up to 80% inhibition by uncouplers only in room light indicate a strong effect of proton-motive force, which can be provided by room light. In addition, unisite ATPase is inhibited by azide as long as some ADP is bound to the CF.

Several differences were found between unisite and multisite ATPase. 1) The unisite activities of both membrane-bound and free enzyme were stimulated up to 3-fold by 4 mM free MgCl (a strong inhibitor of multisite ATPase). 2) Thylakoid unisite ATPase was inhibited by sulfite (50% inhibition at 5 mM), a powerful activator of multisite ATPase. This inhibition is attributed to a nonspecific ionic strength effect. 3) Unisite ATPase was inhibited by trypsin treatment, which increases multisite ATPase severalfold. 4) The pH profile of thylakoid unisite ATPase is somewhat different from that of multisite. 5) Alkylation of Cys-89 of the subunit by N-ethylmaleimide did not affect the unisite activity, but inhibited multisite activity more than 90%.

FOOTNOTES

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This work was supported in part by National Science Foundation Grant DCB-91-11751 (to A. T. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Present address: Molecular Pathophysiology Branch NIDDK, NIH, Building 10, Room 8C-101, 10 Center Dr., MSC 1752, Bethesda, MD 20892-1752.

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To whom correspondence should be addressed: Plant Biology Section, Plant Science Bldg., Cornell University, Ithaca, NY 14853. Tel.: 607-255-8940; Fax: 607-255-5407.

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The abbreviations used are: MF, mitochondrial F; CF, chloroplast coupling factor; CHES, 2-(cyclohexylamino)ethanesulfonic acid; Chl, chlorophyll; DTT, dithiothreitol; EF, E. coli F; MalNEt, N-ethylmaleimide; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; pmf, proton-motive force; PMS, N-methyl phenazonium methosulfate; PSI, photosystem I; PSII, photoystem II; Tricine, N-tris(hydroxymethyl)methylglycine; µE, microeinstein.

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R. E. McCarty, personal communication.

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W. R. Zipfel and S. Zhang, unpublished results.

ACKNOWLEDGEMENTS

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