ORIGINAL ARTICLE William L. Turner Æ Vicki L. Knowles William C. Plaxton Cytosolic pyruvate kinase: subunit composition, activity, and amount in developing castor and soybean seeds, and biochemical characterization of the puriﬁed castor seed enzyme Received: 1 June 2005 / Accepted: 4 June 2005 / Published online: 28 July 2005 Ó Springer-Verlag 2005 Abstract Antibodies against Brassica napus cytosolic pyruvate kinase (PK c ) (EC 2.7.1.40) were employed to examine PK c subunit composition and developmental proﬁles in castor and soybean seeds. A 56-kDa immu- noreactive polypeptide was uniformly detected on im- munoblots of clariﬁed extracts from developing castor endosperm or soybean embryos. Maximal PK c activities occurred early in castor oil seed (COS) and soybean development (7.1 and 5.5 (lmol 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 PK c activity and the relative amount of the immunore- active 56-kDa PK c polypeptide. PK c from developing COS was puriﬁed 1,874-fold to homogeneity and a ﬁnal speciﬁc activity of 73.1 (lmol of pyruvate produced/min) mg 1 protein. Gel ﬁltration and SDS-PAGE indicated that this PK c exists as a 230-kDa homotetramer com- posed of 56-kDa subunits. The mass ﬁngerprint of tryptic peptides of the 56-kDa COS PK c subunit best matched three putative PK c s from Arabidopsis thaliana. The puriﬁed enzyme was relatively heat-stable and dis- played 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 reliev- ing glutamate inhibition. Together with our previous studies, the results: (1) allow a model to be formulated regarding the coordinate allosteric control of PK c and phosphoenolpyruvate carboxylase by aspartate and glutamate in developing COS, and (2) provide further biochemical evidence that castor plant PK c exists as tissue-speciﬁc isozymes that exhibit substantial diﬀer- ences in their respective physical and regulatory prop- erties. Keywords Carbohydrate metabolism Æ Oilseed (developing) Æ phosphoenolpyruvate Æ phospho- enolpyruvate carboxylase (EC 4.1.1.31) Æ Plant glycolysis Æ Pyruvate kinase (EC 2.7.1.40) Æ Glycolytic control Abbreviations COS: Castor oil seed Æ FPLC: Fast protein liquid chromatography Æ MALDI-TOF MS: Laser desorption ionization-time of ﬂight mass spectrometry Æ PEG: Poly(ethylene)glycol Æ PEP: Phosphoenolpyruvate Æ PEPC: Phosphoenolpyruvate carboxylase Æ PK: Pyruvate kinase Æ PK c and PK p : Cytosolic and plastid PK isozymes, respectively. Introduction In developing seeds, the partitioning of imported pho- tosynthate between starch, storage lipid, and storage protein biosynthesis is of major agronomic concern. The metabolism of the glycolytic intermediate phosphoenol pyruvate (PEP) appears to play a prominent role in directing seed carbohydrates towards plastidic fatty acid biosynthesis versus the mitochondrial production of ATP and organic acids required for amino acid inter- conversion in support of storage protein biosynthesis (Ruuska et al. 2002; Schwender et al. 2004). An impor- tant enzyme in glycolytic PEP metabolism is pyruvate kinase (PK; EC 2.7.1.40) which catalyzes the irreversible transfer of Pi from PEP to ADP, yielding pyruvate and ATP. W. L. Turner Æ V. L. Knowles Æ W. C. Plaxton (&) Department of Biology, Queen’s University, Kingston, ON, Canada, K7L 3N6 E-mail: plaxton@biology.queensu.ca URL: http://biology.queensu.ca/faculty/plaxton.html Tel.: +1-613-5336150 Fax: +1-613-5336617 W. C. Plaxton Department of Biochemistry, Queen’s University, Kingston, ON, Canada, K7L 3N6 Planta (2005) 222: 1051–1062 DOI 10.1007/s00425-005-0044-8