Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.
Research Article| December 08 2000
Purification and characterization of cytosolic pyruvate kinase from banana fruit
William L. TURNER;
William C. PLAXTON
William C. PLAXTON 1
*Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6
†Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6
1To whom correspondence should be addressed (e-mail firstname.lastname@example.org).
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William L. TURNER, William C. PLAXTON; Purification and characterization of cytosolic pyruvate kinase from banana fruit. Biochem J 15 December 2000; 352 (3): 875–882. doi: https://doi.org/10.1042/bj3520875
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