Affinity labeling and purification of spinach leaf ribulose-5-phosphate kinase

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Abstract
Spinach leaf ribulose-5-phosphate kinase has been purified to homogeneity by a procedure incorporating affinity chromatography. The purified enzyme requires a divalent cation for activity and has a specific activity of 360 units/mg. It is composed of two apparently identical subunits. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a subunit Mr of 45,000. The enzyme is inactivated by 5''-[p-(fluorosulfonyl)benzoyl]adenosine in a site-directed fashion. The reaction is pseudo first order both in the presence and absence of Mg2+. The presence of Mg2+ retards the nonspecific loss of activity in the absence of the affinity label while accelerating the rate of inactivation by the affinity label. In the presence of Mg2+, Ki = 4.8 mM and kinac+ = 4.22 min-1 at 30.degree. C. The rate of inactivation is slightly accelerated by the presence of ribulose 5-phosphate. While Mg2+-ADP and Mg2+-ATP offer some protection, the greatest protection is provided by Mg2+-ADP-sugar phosphate complexes. The inactivation is largely reversible with dithiothreitol, thus suggesting the modification of an active site cysteine residue.