Activation of sucrose-phosphate synthase from darkened spinach leaves by an endogenous protein phosphatase

Steven C. Huber, Joan L. Huber

Research output: Contribution to journalArticle

Abstract

Sucrose-phosphate synthase (SPS; EC 2.4.1.14) extracted from darkened spinach (Spinacia oleracea L.) leaves has a low activation state, defined as the ratio of activity measured with limiting substrates (plus the inhibitor Pi) to activity with saturating substrates (maximum velocity). Preincubation at 25 °C of desalted crude extracts from darkened leaves resulted in a time-dependent increase in activation state that was inhibited by Pi [IC50 (concentration causing 50% inhibition) ≈3 mm], molybdate, okadaic acid (IC50 ≈ 25 nm) and vanadate, but was stimulated by fluoride. The "spontaneous activation" of SPS in vitro was enhanced slightly by exogenous MgCl2 (up to 5 mm) and exhibited a pH optimum of 7.0 to 7.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with [32P]Pi in the dark was lost with time when extracts were incubated at 25 °C. This loss in radiolabel was substantially reduced by vanadate. These results provide direct evidence for action of an endogenous protein phosphatase(s) using SPS as substrate. The spontaneous activation achieved in vitro could be reversed by subsequent addition of 1 mm Mg · ATP; the activation/ inactivation achieved in vitro was similar in magnitude to the dark-light regulation observed in vivo. Moreover, feeding okadaic acid to excised leaves in the dark blocked subsequent light activation of SPS without affecting photosynthetic rate. These results are consistent with the notion that SPS contains phosphorylation site(s) that reduce enzyme activation state and that dephosphorylation of these residue(s) is the mechanism of light activation. Regulation of the protein phosphatase by Pi may be of physiological significance.

Original languageEnglish (US)
Pages (from-to)421-426
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume282
Issue number2
DOIs
StatePublished - Nov 1 1990

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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