Bot. Mag. Tokyo 84: 159-168 (March 25, 1971) Yoshinori WADA* : Changes in Activities of Ribulosediphosphate Carboxylase and Phosphopyruvate Carboxylase during Leaf Growth of Tobacco. Received September 30, 1970 Abstract 1) Water soluble proteins from leaves of tobacco (N. tabacum Bright Consolation ") and corn (Zea mays) were fractionated by Sephadex G-100 column (2.5 x 40 cm). Fraction 1 protein conteined ribulosediphosphate (RuDP) carboxylase (E. C. 4. 1.1. 39) as well as phosphopyruvate (PP) carboxylase (E. C. 4. 1.1. 31). 2) PP carboxylase was rapidly inactivated when incubated at above 25. On the other hand, RuDP carboxylase was stable and the optimum temperature for the enzyme reaction was located at approximately 40. 3) RuDP carboxylase activity of tobacco leaf on fresh weight basis, attained its maximum when the leaf elongated approximately 50% of its final length, and decreased rapidly thereafter towards leaf senescence. 4) There was a nearly parallel correlation between enzyme activities of RuDP and PP carboxylases and the content of fraction 1 protein of tobacco and corn leaves. 5) The activity of PP carboxylase of young corn leaf was as lower as that of young tobacco leaf, but PP carboxylase activity in matured leaves of corn was 15 times greater than that of tobacco leaves. The activity of RuDP carboxylase was generally lower in corn leaves, 40-70% to tobacco RuDP carboxylase, when compared with the same growth stages of a leaf.
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March, 1971 Ribulosediphosphate Carboxylase and Phosphopyruvate Carboxylase 161 Table 1. Composition of the reaction mixture for the assay of RuDP carboxylase. Table 2. Composition of the reaction mixture for the assay of PP carboxylase. Fig. 1. The elution profile of the water soluble protein fraction of tobacco leaf on a Sephadex G-100 column (2.5 x 45 cm). (a) UV absorption at 280 nm. (b) Enzyme activity. Solid line: carboxylase (E. C. 4.1.1.39). Dotted RuDP line: PP carboxylase (E. C. 4.1.1.31). For the assay of RuDP carboxylase, the reaction mixture in Table 1 was employed. The reaction was stopped by addition of 0.5 ml 6 N acetic acid after 10 minutes incubation at 25. Unreacted HCO3 was removed under reduced pressure. The assay conditions for PP carboxylase were the same as RuDP carboxylase assay except for the component of reaction mixture as shown in Table 2.
162 WADA, 'Y. Vol. 84 Fig. 2. The effect of temperature on the reaction velocity of RuDP carboxylase. After 10 minute (dotted line) or 5 hour (solid line) preincubation at indicated temperatures, enzyme activities were measured at the same temperatures indicated. For other assay conditions, see the legend to Fig. 1. Fig. 3. The effect of HCO3 concentration on the PP carboxylase reaction. The assay conditions were the same as described in the legend to Fig. 1. except for bicarbonate.
March, 1971 Ribulosediphosphate Carboxylase and Phosphopyruvate Carboxylase 163 Fig. 4. Time-course of the PP carboxylase reaction at different temperatures. The assay conditions were the same as in the legend to Fig. 1. Reaction vessels were incubated for the indicated time at 5, 25 sand 50 respectively. Fig. 5. The effect of temperature on the velocity of PP carboxylase reaction. The assay conditions and symbols were the same as in the legend to Fig. 2.
164 WA DA, Y. Vol. 84 Fig. 6. Activities of two carboxylation enzymes (RuDP carboxylase ~I and PP carboxylase LII) and the content of Fraction 1 protein (solid line) in tobacco leaves at different developing stages. Number of leaf age grade indicates, 1 : 2-3 cm in length along midrib (the youngest leaf), 2 : 7-8 cm, 3 : 14-15 cm, 4 : 26-27 cm, 5 : 30-33 cm (fully expanded leaf), 6: 7 day after when the leaf ceases to elongate, 7 : 14 day after when the leaf ceases to elongate. Determination of the enzyme activity was carried out according to the method described in Fig. 1. Fraction 1 was prepared on a Sephadex G-100 column as shown in Fig. 1 and the protein content of the fraction was determined using the method of Lowry et al. * Fresh leaves. Fig. 7. Activities of two carboxylation enzymes and the content of Fraction 1 protein in corn leaves at different growth stages. Symbols were the same as shown in Fig. 6. Grade of leaf age, 1 : Young greenish-yellow leaf, 2 : 20-22 cm in length along midrib, 3 40-45 cm, 4 : Fully elongated leaf, 5 : Matured leaf (lower stalk position), 6 : senescent leaf (old leaf).
March, 1971 Ribulosediphosphate Carboxyl,ase and Phosphopyruvate Carboxylase 165 Fig. 8. Specific different activities growth of the stages. two carboxylation enzymes in Symbols are the, same as used tobacco in Fig. leaves 6. at Fig. 9. Specific activities of the two carboxylation enzymes in corn different growth stages. Symbols are the same as used in Fig. leaves 7. at
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