Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities |
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Authors: | Kent S S Young J D |
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Institution: | Department of Botany and Range Science, Brigham Young University, Provo, Utah 84602. |
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Abstract: | An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase EC 4.1.1.39]. 1-14C,5-3H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate Vc/Vo ratios from the expression A/(B-A) where A and B represent the 3H/14C isotope ratios of doubly labeled RuBP and 3-PGA, and Vc and Vo represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from 2-14C]glucose and 6-3H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase. |
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