The aggregation states of spinach phosphoribulokinase

• Published in: Planta Vol. 181; no. 3; pp. 349 - 357
• Main Author: Porter, M.A. (Oak Ridge National Lab., Tennessee (USA))
• Format: Journal Article
• Language: English
• Published: Berlin Springer-Verlag 01-06-1990; Springer
• Subjects: Aggregation; Biological and medical sciences; Chloroplast; CHLOROPLASTE; CHLOROPLASTS; CLOROPLASTO; DARKNESS; Disulfides; Dunkelheit; Enzymaktivitaet; Enzymes; Filtration; FOSFORILACION; Fundamental and applied biological sciences. Psychology; Gels; Homogenization; Licht; LIGHT; LUMIERE; LUZ; Metabolism; OBSCURIDAD; OBSCURITE; Oxidation; PHOSPHORYLATION; Phosphorylierung; Plant physiology and development; Plants; Spinach; SPINACIA OLERACEA; Spinat; TRANSFERASAS; TRANSFERASE; TRANSFERASES; Light effect; Phosphoribulokinase; Phosphorylation; Spinacia oleracea; Chenopodiaceae; Aggregation; Enzymatic activity; Dicotyledones; Angiospermae; Gel filtration; Plant protein; Spermatophyta; Experimental plant; Chloroplast
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/BF00195887

Phosphoribulokinase (PRK; EC 2.1.7.19) is active in illuminated chloroplasts and inactive in darkened chloroplasts. This regulatory mechanism is mediated by thioredoxin-dependent reduction of a kinase disulfide in vivo. Extracts of spinach (Spinacia oleracea L.) leaves in the presence of 10 mM dithiothreitol contain a single 80-kDa form of PRK as judged by gel filtration. Gel filtration of thiol-free extracts of light-harvested tissue shows the presence of two inactive forms of PRK, the 80-kDa form and an aggregate ( > 550 kDa) form, but treatment of both forms with dithiothreitol restores kinase activity. Gel filtration following extraction of dark-harvested tissue in the absence of dithiotreitol demonstrates the presence of only the heavier form. Inclusion of 400 mM (NH4)2SO4 in the homogenization buffer during extraction of light-harvested tissue suppresses the formation of the high-Mr form of PRK, but does not eliminate the aggregate form observed in extracts of dark-harvested leaves. However, prolonged treatment of extracts from dark-harvested tissue with 400 mM (NH4)2SO4 results in conversion of the high-Mr form of phosphoribulokinase to the low-Mr form. The data are consistent with the heavier form of phosphoribulokinase being the normal in-vivo aggregation state in the dark, while the lighter form is the normal aggregation state in the light.