Abstract
Complete stoichiometry of the reaction catalyzed by ribulose 1,5-bisphosphate (RuBP) oxygenase from spinach and Rhodospirillum rubrum has been determined. Before initiation and after termination, RuBP has been measured either by release of equimolar orthophosphate at 25°C in the presence of 1 n NaOH or by complete carboxylation using 14CO2 and RuBP carboxylase. The RuBP-dependent oxygen consumption has been measured continuously with an oxygen electrode. After termination of catalysis, 3-phosphoglycerate production has been determined spectrophotometrically using phosphoglycerokinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, α-glycerophosphate dehydrogenase, ATP, and NADH. To measure phosphoglycolate, this product was first hydrolyzed with alkaline phosphatase and the resultant glycolate oxidized by glycolate oxidase. Attendant H2O2 formation catalyzed by peroxidase has then been measured colorimetrically. Interference by ribulose in the measurement of glycolate can be easily corrected. Procedures are rapid and do not require separation of reactants and products. Results are in excellent accord with the expected stoichiometry for catalysis by RuBP oxygenase and also enable an estimate of competing catalysis by RuBP carboxylase.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 158-166 |
| Number of pages | 9 |
| Journal | Analytical Biochemistry |
| Volume | 124 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jul 15 1982 |
| Externally published | Yes |
Bibliographical note
Funding Information:’ Supported in part by Grant GM 19,972 from the NIH. ’ Present address: Nabisco Brands, Wilton, Connecticut 06897. 3 To whom inquiries should be addressed. ’ Present address: Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, New York 14853. ‘Abbreviations used: RuBP, D-ribulose l,%bisphosphate; 3-PGA, 3-phospho-D-glycerate; P-glycolate, phosphoglycolate.