Development and Characterization of Ribulose-1,5-bisphosphate Carboxylase

Ribulose-1,5-bisphosphate carboxylase activity was found in endosperm of germinating castor bean seed Ricinus communis and was localized in proplastids. The endosperm carboxylase has been extensively purified and is composed of two different subunits. The molecular weights of the native carboxylase and its subunits were 560,000, 55,000, and 15,000 daltons, respectively. The Michaelis-Menten constants, K(m), for the endosperm carboxylase with respect to ribulose 1,5-bisphosphate, bicarbonate, CO(2), and magnesium in millimolar are 0.54, 13.60, 0.92, and 0.57, respectively. The endosperm carboxylase was activated by Mg(2+) and HCO(3) (-). The preincubation of the carboxylase with 1 millimolar HCO(3) (-) and 5 millimolar MgCl(2) resulted in activation by low and inhibition by high concentrations of 6-phosphogluconate.In studies of dark (14)CO(2) fixation by endosperm slices, [(14)C]malate and [(14)C]citrate were the predominantly labeled products after 30 seconds of exposure of the tissue to H(14)CO(3) (-). In pulse-chase experiments, 87% of the label is malate, and citrate was transferred to sugars after a 60-minute chase with a small amount of the label appearing in the incubation medium as (14)CO(2). The minimal incorporation of the label from (14)CO(2) into phosphoglyceric acid indicated a lack of the endosperm ribulose-1,5-bisphosphate carboxylase participation in the endosperm's CO(2) fixation system. The activities of key Calvin cycle enzymes were examined in the endosperms and cotyledons of dark-grown castor bean seedlings. Many of these autotrophic enzymes develop in the dark in these tissues. The synthesis of ribulose-1,5-bisphosphate carboxylase in the nonphotosynthetic endosperms is not repressed in the dark, and high levels of enzymic activity appear with germination. All of the Calvin cycle enzymes are present in the castor bean endosperm except NADP-linked glyceraldehyde 3-P dehydrogenase, and the absence of this dehydrogenase probably prevents the functioning of these series of reactions in dark CO(2) fixation.