Affinity labeling and purification of spinach leaf ribulose-5-phosphate kinase.
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 dodecylsulfate-polyacrylamide gel electrophoresis indicates a subunit M, 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 Mg(2+). The presence of Mg(2+) 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 Mg(2+),K (i) = 4.8 mM and k(inact) = 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 Mg(2+)-ATP offer some protection, the greatest protection is provided by Mg(2+)-ADP-sugar phosphate complexes. The inactivation is largely reversible with dithiothreitol, thus suggesting the modification of an active site cysteine residue.
Krieger
T J
TJ
Miziorko
H M
HM