skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

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 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.

Authors:
; ; ;
Publication Date:
Research Org.:
Oak Ridge National Lab., TN
OSTI Identifier:
5734603
Resource Type:
Journal Article
Journal Name:
Arch. Biochem. Biophys.; (United States)
Additional Journal Information:
Journal Volume: 1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 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

Citation Formats

Porter, M A, Milanez, S, Stringer, C D, and Hartman, F C. Purification and characterization of ribulose-5-phosphate kinase from spinach. United States: N. p., 1986. Web. doi:10.1016/0003-9861(86)90185-2.
Porter, M A, Milanez, S, Stringer, C D, & Hartman, F C. Purification and characterization of ribulose-5-phosphate kinase from spinach. United States. https://doi.org/10.1016/0003-9861(86)90185-2
Porter, M A, Milanez, S, Stringer, C D, and Hartman, F C. 1986. "Purification and characterization of ribulose-5-phosphate kinase from spinach". United States. https://doi.org/10.1016/0003-9861(86)90185-2.
@article{osti_5734603,
title = {Purification and characterization of ribulose-5-phosphate kinase from spinach},
author = {Porter, M A and Milanez, S and Stringer, C D and Hartman, F C},
abstractNote = {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.},
doi = {10.1016/0003-9861(86)90185-2},
url = {https://www.osti.gov/biblio/5734603}, journal = {Arch. Biochem. Biophys.; (United States)},
number = ,
volume = 1,
place = {United States},
year = {Sat Feb 15 00:00:00 EST 1986},
month = {Sat Feb 15 00:00:00 EST 1986}
}