FACTORS INFLUENCING THE MODE OF ENERGY COUPLING IN THYLAKOID MEMBRANES

WILLIAM ALBERT BEARD, Purdue University

Abstract

The luciferin-luciferase ATP detection system allows for a simple and sensitive assay to follow ATP formation initiated by single-turnover flashes. This assay can be used to determine the number of flashes to reach the energetic threshold as well as detecting post-illumination ATP formation after the last flash in a flash sequence. Post-illumination phosphorylation can be observed after a series of phosphorylating flashes (PIP('+)) or when the assay for ATP formation is performed in a 'traditional' manner where the ADP and P(,i) are added after the last flash of a flash train (PIP('-)). It was found that PIP('+) was stimulated by the presence of valinomycin-K('+), by decreasing the external pH, and increasing the flash frequency. When ADP and P(,i) were added to the thylakoid suspension after a train of flashes, the PIP('-) ATP yield was influenced by pyridine as expected, in a manner consistent with ATP formation, in part, being driven by protons present in the bulk inner aqueous phase. However, when ADP and P(,i) were present during the flash train, the PIP('+) ATP yield was unaffected by the presence of pyridine. The results support the hypothesis that electron transfer-released protons can energize ATP formation driven by localized proton gradients associated with endogenous membrane phase buffering groups. When 100 mM KCl replaced sucrose in the thylakoid resuspension buffer, thylakoids were converted from a localized energy coupling mode to a bulk phase delocalized coupling mode as determined by the sensitivity of the onset of ATP formation and the PIP('+) ATP yield to pyridine. Thylakoids isolated in the presence of KCl also exhibited a lag for the onset of ATP formation which was about two times longer than sucrose-isolated thylakoids. These results, in addition to proton uptake measurements determined with cresol red, indicated that the size of the threshold buffering pool was larger for thylakoids isolated in the presence of KCl. Since pyridine increased the size of the threshold buffering pool with KCl-isolated thylakoids, this pool was probably composed of buffering groups which were in the lumen. These buffering groups, however, were not important in reaching the energetic thresholds when thylakoids were isolated in the absence of KCl. (Abstract shortened with permission of author.)

Degree

Ph.D.

Subject Area

Biochemistry

COinS