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Plant Physiol. Feb 1981; 67(2): 237–245.
PMCID: PMC425662

A Sensitive Assay Procedure for Simultaneous Determination of Ribulose-1,5-bisphosphate Carboxylase and Oxygenase Activities

Abstract

A sensitive assay procedure is described for the simultaneous determination of ribulose-1,5-bisphosphate (RuBP) carboxylase and oxygenase activities. In this assay, [1-3H]RuBP is incubated with 14CO2 and O2. Carboxylation rate is determined from 14CO2 incorporation and oxygenation rate is determined from [2-3H]glycolate-phosphate production. The assay was found to be suitable at all CO2 and O2 concentrations examined, which ranged from 0 to 300 micromolar CO2 (20 millimolar NaHCO3) and 0 to 1.15 millimolar (100%) O2. In combination with a polarographic assay, the stoichiometry of the RuBP oxygenase reaction was found to be RuBP-O2-glycolate phosphate-glycerate phosphate (1:1:1:1).

The ratio of soybean (Merr.) RuBP carboxylase and oxygenase activities was measured at various CO2 and O2 concentrations and in the presence of several compounds which have been reported to alter differentially the rate of the two reactions. The ratio of the two activities was a linear function of the ratio of the CO2 and O2 concentrations. The substrate specificity factor VcKo/VoKc, which determines relative carboxylase and oxygenase reaction rates as a function of the CO2 and O2 concentrations, was found to be 73 in the presence of Mg2+. Of the several compounds which have been reported to alter differentially the two reactions, we found that only Mn2+ substitution for Mg2+ was effective. Compared to Mg2+, Mn2+ reduced the Km(O2) from 690 to 36 micromolar O2 and reduced the specificity factor to about 4. The Km(CO2) was about 20 micromolar CO2 in the presence of both Mg2+ and Mn2+. Comparison of reaction rates in the presence of activated and inactivated enzyme allowed a direct determination that both carboxylase and oxygenase activities are similarly activated by CO2, with an activation equilibrium constant of about 1.3 millimolar NaHCO3 (27 micromolar CO2) at pH 7.85 and 10 millimolar Mg2+.

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Selected References

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