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. 1980 Mar;65(3):465–468. doi: 10.1104/pp.65.3.465

Simultaneous Kinetic Analysis of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activities 1

Samuel S Kent 1,2, Joseph D Young 1
PMCID: PMC440355  PMID: 16661214

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.

The kinetic properties of a commercial preparation of fully activated spinach carboxylase were studied under approximated physiological conditions of 20% O2 (252 micromolar), 295 μl/l CO2 (10 micromolar), 25 C, and pH 8.19. The Vc/Vo ratio was, within experimental error, constant at 30 seconds and 1 minute. This double label assay method may be used to calculate Vc/Vo ratios for the Laing-Ogren-Hageman equation, Vc/Vo = (VcKo/VoKc) ([CO2]/[O2]) where Vc and Vo represent Vmax, and Kc and Ko represent Michaelis constants for the carboxylase and oxygenase activities, respectively.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrews T. J., Badger M. R., Lorimer G. H. Factors affecting interconversion between kinetic forms of ribulose diphosphate carboxylase-oxygenase from spinach. Arch Biochem Biophys. 1975 Nov;171(1):93–103. doi: 10.1016/0003-9861(75)90011-9. [DOI] [PubMed] [Google Scholar]
  2. Andrews T. J., Lorimer G. H., Tolbert N. E. Ribulose diphosphate oxygenase. I. Synthesis of phosphoglycolate by fraction-1 protein of leaves. Biochemistry. 1973 Jan 2;12(1):11–18. doi: 10.1021/bi00725a003. [DOI] [PubMed] [Google Scholar]
  3. Badger M. R., Andrews T. J. Effects of CO2, O2 and temperature on a high-affinity form of ribulose diphosphate carboxylase-oxygenase from spinach. Biochem Biophys Res Commun. 1974 Sep 9;60(1):204–210. doi: 10.1016/0006-291x(74)90192-2. [DOI] [PubMed] [Google Scholar]
  4. Bowes G., Ogren W. L., Hageman R. H. Phosphoglycolate production catalyzed by ribulose diphosphate carboxylase. Biochem Biophys Res Commun. 1971 Nov 5;45(3):716–722. doi: 10.1016/0006-291x(71)90475-x. [DOI] [PubMed] [Google Scholar]
  5. Bowes G., Ogren W. L. Oxygen inhibition and other properties of soybean ribulose 1,5-diphosphate carboxylase. J Biol Chem. 1972 Apr 10;247(7):2171–2176. [PubMed] [Google Scholar]
  6. Keck R. W. Differential Oxygen Response of Photosynthesis in Soybean and Panicum milioides. Plant Physiol. 1976 Oct;58(4):552–555. doi: 10.1104/pp.58.4.552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Krenzer E. G., Moss D. N., Crookston R. K. Carbon dioxide compensation points of flowering plants. Plant Physiol. 1975 Aug;56(2):194–206. doi: 10.1104/pp.56.2.194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Laing W. A. Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase. Plant Physiol. 1974 Nov;54(5):678–685. doi: 10.1104/pp.54.5.678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lorimer G. H., Andrews T. J., Tolbert N. E. Ribulose diphosphate oxygenase. II. Further proof of reaction products and mechanism of action. Biochemistry. 1973 Jan 2;12(1):18–23. doi: 10.1021/bi00725a004. [DOI] [PubMed] [Google Scholar]
  10. Lorimer G. H., Badger M. R., Andrews T. J. D-Ribulose-1,5-bisphosphate carboxylase-oxygenase. Improved methods for the activation and assay of catalytic activities. Anal Biochem. 1977 Mar;78(1):66–75. doi: 10.1016/0003-2697(77)90009-4. [DOI] [PubMed] [Google Scholar]
  11. Lorimer G. H., Badger M. R., Andrews T. J. The activation of ribulose-1,5-bisphosphate carboxylase by carbon dioxide and magnesium ions. Equilibria, kinetics, a suggested mechanism, and physiological implications. Biochemistry. 1976 Feb 10;15(3):529–536. doi: 10.1021/bi00648a012. [DOI] [PubMed] [Google Scholar]
  12. Marsho T. V., Kung S. D. Oxygenase properties of crystallized fraction 1 protein from tobacco. Arch Biochem Biophys. 1976 Mar;173(1):341–346. doi: 10.1016/0003-9861(76)90268-x. [DOI] [PubMed] [Google Scholar]
  13. McCurry S. D., Tolbert N. E. Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by xylulose 1,5-bisphosphate. J Biol Chem. 1977 Dec 10;252(23):8344–8346. [PubMed] [Google Scholar]
  14. Moss D. N., Krenzer E. G., Jr, Brun W. A. Carbon dioxide compensation points in related plant species. Science. 1969 Apr 11;164(3876):187–188. doi: 10.1126/science.164.3876.187. [DOI] [PubMed] [Google Scholar]
  15. Nishimura M., Graham D., Akazawa T. Effect of oxygen on photosynthesis by spinach leaf protoplasts. Plant Physiol. 1975 Nov;56(5):718–722. doi: 10.1104/pp.56.5.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ogren W. L., Hunt L. D. Comparative biochemistry of ribulose bisphosphate carboxylase in higher plants. Basic Life Sci. 1978;11:127–128. doi: 10.1007/978-1-4684-8106-8_9. [DOI] [PubMed] [Google Scholar]
  17. Ryan F. J., Barker R., Tolbert N. E. Inhibition of ribulose diphosphate carboxylase/oxygenase by xylitol 1,5-diphosphate. Biochem Biophys Res Commun. 1975 Jul 8;65(1):39–46. doi: 10.1016/s0006-291x(75)80058-1. [DOI] [PubMed] [Google Scholar]
  18. Servaites J. C. Oxygen inhibition of photosynthesis and stimulation of photorespiration in soybean leaf cells. Plant Physiol. 1978 Jan;61(1):62–67. doi: 10.1104/pp.61.1.62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Servaites J. C. pH Dependence of Photosynthesis and Photorespiration in Soybean Leaf Cells. Plant Physiol. 1977 Nov;60(5):693–696. doi: 10.1104/pp.60.5.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. WEISSBACH A., HORECKER B. L., HURWITZ J. The enzymatic formation of phosphoglyceric acid from ribulose diphosphate and carbon dioxide. J Biol Chem. 1956 Feb;218(2):795–810. [PubMed] [Google Scholar]
  21. Wishnick M., Lane M. D. Inhibition of ribulose diphosphate carboxylase by cyanide. Inactive ternary complex of enzyme, ribulose diphosphate, and cyanide. J Biol Chem. 1969 Jan 10;244(1):55–59. [PubMed] [Google Scholar]

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