Purification and characterization of ribulose-5-phosphate kinase from spinach
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 Mr 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 NH2-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.
- Research Organization:
- Oak Ridge National Lab., TN
- OSTI ID:
- 5734603
- Journal Information:
- Arch. Biochem. Biophys.; (United States), Vol. 1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
PHOSPHOTRANSFERASES
MOLECULAR STRUCTURE
AMINO ACIDS
CHLOROPLASTS
CHROMATOGRAPHY
MOLECULAR WEIGHT
PLANTS
PURIFICATION
SPINACH
CARBOXYLIC ACIDS
CELL CONSTITUENTS
ENZYMES
FOOD
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
SEPARATION PROCESSES
TRANSFERASES
VEGETABLES
550200* - Biochemistry