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Use of phosphopeptides to study thylakoid protein phosphatase activity
Abstract
Thylakoid protein phosphatase is involved in the regulation of light energy distribution between the two photosystems of photosynthesis. Efforts to investigate the thylakoid protein phosphatase activity have been hindered by the lack of a suitable substrate. Phosphohistone and phosphorylase a, substrates for most protein phosphatase activities, are not dephosphorylated by this enzyme. Specific inhibitors for protein phosphatase 1 and 2A, microcystin-LR and okadaic acid, do not inhibit the thylakoid protein phosphatase. These results suggest that the thylakoid protein phosphatase may not fit into the current classification of protein phosphatase activities. Pea chloroplast has no phosphatase activity that hydrolyzes 4-methylumbelliferyl phosphate, b-glycerophosphate, or several small phosphopeptides, all of which are alkaline phosphatase substrates. These results demonstrate that significant alkaline phosphatase activity is not present in the chloroplast and therefore can not be responsible for the dephosphorylation of thylakoid phosphoproteins. Phosphopeptides mimicking the phosphorylation site of the major thylakoid phosphoprotein, light-harvesting chlorophyll a/b-binding protein, was dephosphorylated by pea thylakoid membranes. The dephosphorylation was similar to the dephosphorylation of endogenous light-harvesting chlorophyll a/b-binding protein in dephosphorylation rate, sensitivity to inhibitors, pH dependence profile, and divalent cation requirements. Furthermore, the thylakoid membrane dephosphorylates only the phosphopeptide mimicking the light-harvesting chlorophyll a/b-binding protein, not any other tested. These similarities between the dephosphorylation of phosphopeptide and the light harvesting chlorophyll a/b-binding protein and the strict selection of the light-harvesting chlorophyll a/b-binding protein sequences by the thylakoid phosphopeptide phosphatase suggest that both activities are catalyzed by the same enzyme. Phospho-light-harvesting chlorophyll a/b-binding protein peptide markedly inhibited endogenous light-harvesting chlorophyll a/b-binding protein dephosphorylation while the unphosphorylated peptide had no effect. Furthermore, only those phosphopeptide sequences that could be dephosphorylated by thylakoid membranes inhibited light-harvesting chlorophyll a/b-binding protein dephosphorylation. Collectively these results demonstrate that phosphopeptides mimicking the phosphorylation site of a thylakoid phosphoprotein can be used to study the thylakoid protein phosphatase.
Subject Area
Biochemistry|Botany|Biology
Recommended Citation
Sun, Gongqin, "Use of phosphopeptides to study thylakoid protein phosphatase activity" (1992). ETD collection for University of Nebraska-Lincoln. AAI9308198.
https://digitalcommons.unl.edu/dissertations/AAI9308198