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

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Abstract
Ribulose-1,5-bisphosphate carboxylase activity was found in endosperm of germinating castor bean seed R. communis [cv. Hale and cimarron], and was localized in proplastids. The endosperm carboxylase was extensively purified and was composed of 2 different subunits. The MW of the native carboxylase and its subunits were 560,000, 55,000 and 15,000 daltons, respectively. The Michaelis-Menten constants, Km, for the endosperm carboxylase with respect to ribulose 1,5-bisphosphate, bicarbonate, CO2, and Mg were 0.54, 13.60, 0.92 and 0.57 mM, respectively. The endosperm carboxylase was activated by Mg2+ and HCO3-. The preincubation of the carboxylase with 1 mM HCO3- and 5 mM MgCl2 resulted in activation by low and inhibition by high concentrations of 6-phosphogluconate. In studies of dark 14CO2 fixation by endosperm slices, [14C]malate and[14C]citrate were the predominant labeled products after 30 s of exposure of the tissue to H14CO2-. In pulse-chase experiments, 87% of the label is malate and citrate was transferred to sugars after a 60-min chase with a small amount of the label appearing in the incubation medium as 14CO2. The minimal incorporation of the label from 14CO2 into phosphoglyceric acid indicated a lack of the endosperm ribulose-1,5-bisphosphate carboxylase participation in the endosperm''s CO2 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 CO2 fixation.