PROTEIN KINASE STUDIES IN CHLOROPLAST THYLAKOID MEMBRANES (ELECTRON TRANSPORT, FLUOROSULFONYLBENZOYL ADENOSINE, FLUORESCENCE, PURIFICATION)
Abstract
Phosphorylation of spinach chloroplast membranes caused a decrease of approximately 15% in the rates of photosystem II and an equal but opposite stimulation of photosystem I partial reactions under limiting light conditions. No effect of phosphorylation on electron transport was observed at saturating light intensity. These effects could only be measured accurately after minimizing the photoinhibition of electron transport that resulted from the illumination used to activate the chloroplast protein kinase. Treatment of spinach thylakoids with the adenosine affinity inhibitor 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) resulted in (GREATERTHEQ)95% inhibition of phosphorylation of the light harvesting protein complex of photosystem II (LHC-II), while the M(,r) 10,000 polypeptide showed a 35% decrease in phosphorylation. ('14)C FSBA labelled several polypeptides, but only one of M(,r) 50,000 showed significant protection against the label by prior addition of ADP or adenosine, making it a possible candidate for the LHC-II kinase. FSBA had no effect on electron transport and ('14)C FSBA did not label LHC-II, indicating that the FSBA was not interfering with kinase activation or modifying substrates, but rather acting at the level of the LHC-II kinase. Phosphorylation of the M(,r) 10,000 polypeptide subsequent to FSBA treatment required light or reducing conditions in the dark. Inhibition of LHC-II phosphorylation by FSBA eliminated the slow ATP-induced decrease in variable fluorescence. This provides evidence for the direct involvement of LHC-II in the redistribution of excitation energy as the result of phosphorylation. The M(,r) 10,000 phosphoprotein was purified from photosytem II particles by solubilizing the particles in 5% (w/v) dodecyl dimethylamine oxide, centrifugation in 10% (w/v) sucrose, and three column chromatography steps. The purified protein showed a unique NH(,2)-terminus indicating a highly purified polypeptide. The amino acid sequence for the first nine residues is NH(,2)-Ala-Thr-Gln-Thr-Val-Glu-Ser-Ser-Ser....COOH. The amino acid composition was determined and could also be used to help distinguish the polypeptide from other known thylakoid proteins. The sequence and composition data indicated that the M(,r) 10,000 phosphoprotein is neither the hydrophobic protein CF(,0) or cytochrome b-559, but rather a unique, as yet unidentified, polypeptide associated with photosystem II.
Degree
Ph.D.
Subject Area
Biochemistry
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