USE OF ACETIC ANHYDRIDE AS A CHEMICAL MODIFICATION PROBE FOR PHOTOSYSTEM-SPECIFIC PROTON-MEMBRANE INTERACTION IN CHLOROPLASTS

GARY MICHAEL BAKER, Purdue University

Abstract

Acetic anhydride is a protein chemical modifier that reacts preferentially with nonprotonated primary amine groups, such as the (epsilon)-amino group of lysine. Treatment of dark-maintained thylakoid membranes with acetic anhydride shows no effect on subsequent electron transport activity unless measures are taken to increase membrane proton permeability. Uncoupler addition, a brief thermal exposure, or removal of extrinsic coupling factor promotes 40--80% inhibition of water oxidation activity by the anhydride, and leads to greater incorporation of acetyl units into total membrane protein. These results indicate the occurrence of a metastable proton gradient in dark-adapted membranes (measured to ('(TURN))20-35 nmol H('+) mg chl('-1)) that maintains the protonated state of amine groups, some critical for water splitting function. This gradient is shown to reduce the onset time for presteady state ATP synthesis when compared to a proton-depleted (anhydride reactive) control case. Electron transport conditions protect against the inhibition of water oxidation activity by the anhydride, and correspondingly give lower acetyl binding to total membrane protein. The extent of light protection by either photosystem depends on the magnitude of the redox-coupled proton accumulation. These results suggest that anhydride-detectable amine groups are accessible to protons from either protolytic reaction, and conflict with earlier conclusions of Prochaska and Dilley (1978a,b,c). A possible explanation is given in the text.

Degree

Ph.D.

Subject Area

Biology

COinS