Cytosolic pyruvate kinase: subunit composition, activity, and amount in developing castor and soybean seeds, and biochemical characterization of the purified castor seed enzyme
2005
Turner, W.L. | Knowles, V.L.
Antibodies against Brassica napus cytosolic pyruvate kinase (PKc) (EC 2.7.1.40) were employed to examine PKc subunit composition and developmental profiles in castor and soybean seeds. A 56-kDa immunoreactive polypeptide was uniformly detected on immunoblots of clarified extracts from developing castor endosperm or soybean embryos. Maximal PKc activities occurred early in castor oil seed (COS) and soybean development (7.1 and 5.5 (μmol of pyruvate produced/min) g-¹ FW, respectively) and were up to 25-fold greater than those of fully mature seeds. Time-course studies revealed a close correlation between extractable PKc activity and the relative amount of the immunoreactive 56-kDa PKc polypeptide. PKc from developing COS was purified 1,874-fold to homogeneity and a final specific activity of 73.1 (μmol of pyruvate produced/min) mg-¹ protein. Gel filtration and SDS-PAGE indicated that this PKc exists as a 230-kDa homotetramer composed of 56-kDa subunits. The mass fingerprint of tryptic peptides of the 56-kDa COS PKc subunit best matched three putative PKcs from Arabidopsis thaliana. The purified enzyme was relatively heat-stable and displayed a broad pH optimum of 6.4. However, more efficient substrate utilization (in terms of V max /K m for phosphoenolpyruvate or ADP) was observed at pH 7.4. Glutamate was the most effective inhibitor, whereas aspartate functioned as an activator by partially relieving glutamate inhibition. Together with our previous studies, the results: (1) allow a model to be formulated regarding the coordinate allosteric control of PKc and phosphoenolpyruvate carboxylase by aspartate and glutamate in developing COS, and (2) provide further biochemical evidence that castor plant PKc exists as tissue-specific isozymes that exhibit substantial differences in their respective physical and regulatory properties.
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