Purification and Characterization of Phosphoribulokinase from Immature Pods of Brassica

Phosphoribulokinase (ATP:D — ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19; PRuK) from immature pods of Brassica was purified to apparent homogeneity with about 31% recovery using ammonium sulphate fractionation, gel filtration through Sepharose CL-6B and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme, having molecular mass of about 180 kD, was heterotetramer with subunit molecular mass of 48, 47, 41 and 33 kD. The enzyme had an absolute requirement for a divalent cation Mg²⁺ and a monovalent cation K⁺for optimal activity. At optimum pH of 8.0–8.4, the enzyme showed typical hyperbolic response for both the substrates with Km values of 333 μM and 100 μM, respectively for Ru5P and ATP. The enzyme was inhibited by RU-1, 5-P₂, 6-phosphogluconate and AMP, and activatded by glu-1-P, glu-6-P and Pl. RU-1, 5-P₂ and 6-phosphogluconate inhibited the enzyme competitively with respect to Ru5P and non-competitively with respect to ATP. It appears that the activity of the Brassica pod enzyme besides being controlled at the level of metabolites, is regulated by light and energy status of the cell.