Purification and characterization of cytosolic pyruvate kinase from leaves of the castor oil plant.
Cytosolic pyruvate kinase (PKc) from leaves of the castor oil plant (Ricinus communis L.) has been purified 3900-fold to apparent homogeneity and a final specific activity of 51 mumol of pyruvate produced/min/mg protein. PAGE, immunoblot, and gel filtration analyses of the final preparation indicated that this enzyme is an alpha 2 beta 2 heterotetramer of about 250 kDa that is composed of an equivalent ratio of 57- and 56-kDa subunits. The enzyme was relatively heat-stable and displayed a broad pH optimum of approximately 6.5. However, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] occurred at pH 7.5. Enzyme activity was absolutely dependent upon the simultaneous presence of bivalent and a univalent metal cation, with Mg2+ and K+ fulfilling this requirement. Hyperbolic saturation kinetics were observed with PEP, ADP, and K+, whereas Mg2+ binding exhibited positive cooperativity. Mg2 citrate, oxalate, and glutamate were the most effective inhibitors at pH 7.5. Inhibition by these compounds was more pronounced at pH 7.5 than at pH 6.5 and they yielded additive inhibition when tested in pairs. Aspartate functioned as an activator by facilitating the binding of PEP and relieving the inhibition of PKc by glutamate. The in vivo activity of leaf PKc is probably regulated by the relative cytosolic levels of citrate, glutamate, and aspartate. This provides a possible rationale for the known activation of leaf PKc that occurs during periods of enhanced ammonia assimilation. Together with our previous studies, the results also indicate that castor oil plant PKc exists as tissue-specific isoforms that demonstrate substantial differences in their respective physical and/or kinetic and regulatory properties.
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Department of Biology, Queen's University, Kingston, Ontario, Canada.
Plaxton
W C
WC
D000818:Animals; D000906:Antibodies; D002367:Ricinus communis / Q000201:enzymology*; D003429:Cross Reactions; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006019:Glycolysis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010947:Plants, Toxic; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D014018:Tissue Distribution
D000906:Antibodies; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011770:Pyruvate Kinase