ASSESSING MODULATION OF STROMAL AND THYLAKOID LIGHT-HARVESTING COMPLEX-II PHOSPHATASE-ACTIVITIES WITH PHOSPHOPEPTIDE SUBSTRATES

The study of the light-harvesting complex II (LHC-II) phosphatase acti vity has been difficult due to the membrane association of its substra te. Thylakoid membranes labeled with [gamma-P-32]ATP were incubated wi th chymotrypsin, releasing phosphopeptides which served as labeled sub strates for LHC-II phosphatase. Utilizing these phosphopeptides as sub strates, protein phosphatase activities have been identified in both t he thylakoid membrane and the stromal fraction. The thylakoid-bound ph osphatase was liberated from the membrane with a sub-solubilizing conc entration of Brij 35. The membrane and the stromal protein phosphatase s were inhibited by NaF and EDTA, but not inhibited by microcystin-LR. The stromal phosphatase differed from the membrane phosphatase in pH optimum, in its lack of inhibition by molybdate ions, and by its respo nse to magnesium and manganese ions. Using the soluble chymotryptic pe ptide substrate, the effect of light on pea thylakoid-bound LHC-II pho sphatase activity was also assessed. Incubation of the thylakoid membr anes in the light caused a 35% inhibition of LHC-II phosphatase activi ty. The inhibition was diminished by the addition of DCMU. Addition of 10 mM dithiothreitol stimulated the activity in darkness and obviated the inhibition when exposed to light. These studies suggest that posi tive or negative regulation of the LHC-II phosphatase activity is poss ible in vivo.