Assessing modulation of stromal and thylakoid light-harvesting complex-II phosphatase activities with phosphopeptide substrates

Mark F. Hammer, Gautam Sarath, John C. Osterman, John Markwell

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

The study of the light-harvesting complex II (LHC-II) phosphatase activity has been difficult due to the membrane association of its substrate. Thylakoid membranes labeled with [γ-32P]ATP were incubated with chymotrypsin, releasing phosphopeptides which served as labeled substrates for LHC-II phosphatase. Utilizing these phosphopeptides as substrates, protein phosphatase activities have been identified in both the thylakoid membrane and the stromal fraction. The thylakoid-bound phosphatase was liberated from the membrane with a sub-solubilizing concentration of Brij 35. The membrane and the stromal protein phosphatases 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 response to magnesium and manganese ions. Using the soluble chymotryptic peptide substrate, the effect of light on pea thylakoid-bound LHC-II phosphatase activity was also assessed. Incubation of the thylakoid membranes in the light caused a 35% inhibition of LHC-II phosphatase activity. 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 positive or negative regulation of the LHC-II phosphatase activity is possible in vivo.

Original languageEnglish (US)
Pages (from-to)107-115
Number of pages9
JournalPhotosynthesis Research
Volume44
Issue number1-2
DOIs
StatePublished - May 1995

Keywords

  • chloroplast
  • light-harvesting complex
  • protein phosphatase
  • protein phosphorylation
  • thylakoid membrane

ASJC Scopus subject areas

  • Biochemistry
  • Plant Science
  • Cell Biology

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