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Purification and characterization of ribulose-5-phosphate kinase from spinach

Authors
Journal
Archives of Biochemistry and Biophysics
0003-9861
Publisher
Elsevier
Publication Date
Volume
245
Issue
1
Identifiers
DOI: 10.1016/0003-9861(86)90185-2
Keywords
  • Plant Biochemistry And Photosynthesis
Disciplines
  • Biology

Abstract

Abstract An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44, 000 M r subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH 2-terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation.

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