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Metabolism and Enzymology

Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds ¹

Characterization of the Enzyme's Degradation by a Cysteine Endopeptidase

Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada

Leucoplast pyruvate kinase (PK_p; EC 2.7.1.40) from endosperm of developing castor oil seeds (Ricinus communis L. cv Baker 296) appears to be highly susceptible to limited degradation by a cysteine endopeptidase during the purification of the enzyme or incubation of clarified homogenates at 4°C. Purified castor seed PK_p was previously reported to consist of immunologically related 57.5 and 44 kilodalton subunits (Plaxton WC, Dennis DT, Knowles VL [1990] Plant Physiol 94: 1528-1534). By contrast, immunoreactive polypeptides of about 63.5 and 54 kilodaltons were observed when a western blot of an extract prepared under denaturing conditions was probed with affinity purified rabbit anti-(castor seed PK_p) immunoglobulin G. Proteolytic activity against PK_p was estimated by the disappearance of the 63.5 and 54 kilodalton subunits and the concomitant appearance of lower molecular mass immunoreactive degradation products during the incubation of clarified homogenates at 4°C. The presence of 2 millimolar dithiothreitol accelerated the degradation of PK_p. The conservation of the 63.5 and 54 kilodalton subunits was observed after extraction of the enzyme in the presence of 1 millimolar p-hydroxymecuribenzoate, or 1 millimolar N-p-tosyl-L-lysine chloromethyl ketone, or 10 millimolar iodoacetate. These results reveal that a cysteine endopeptidase was responsible for the in vitro proteolysis of PK_p. This endopeptidase is present throughout all stages of endosperm development. Its PK_p-degrading activity, however, appears to be most pronounced in preparations from older endosperm. When lysates of purified leucoplasts were incubated at 4°C for up to 21 hours, no degradation of PK_p was observed; this indicated an extra-leucoplastic localization for the cysteine endopeptidase. Although the in vivo subunit structure of PK_p remains uniform throughout all stages of endosperm development, the large decrease in PK activity that accompanies castor seed maturation coincides with a marked reduction in the concentration of PK_p.

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