Protein purification, assessment of purity, quantitation and assays for determining kinetic constants were done as described elsewhere [31 (link), 33 (link)]. Tritium-labeled RuBP, which was used for measuring CO2/O2 specificity factors, had been synthesized in the laboratory previously [31 (link)]. PRK activities were measured in cell extracts by the addition of NaH14CO3 (Perkin-Elmer), ribulose-5-phosphate (Sigma) and ATP (Sigma) as substrates, and coupling the reaction with excess RubisCO. All kinetic constants measured with either the histidine-tagged megaplasmid RubisCO (Table 2 ) or the untagged chromosomal version of the wild type and A380V single-mutant proteins (Table 3 ) were similar.
Ribulose 5 phosphate
Manufactured by Merck Group
Sourced in Ukraine
Ribulose-5-phosphate is a chemical compound that serves as an intermediate in the Calvin cycle of photosynthesis. It is a key substrate in the conversion of carbon dioxide to organic compounds within plant cells.
Automatically generated - may contain errors
Lab products found in correlation
Ribose 5 phosphate, by Merck Group (3 mentions)
Dihydroxyacetone phosphate, by Merck Group (2 mentions)
Glucose 6 phosphate (g6p), by Roche (2 mentions)
Mannose 6 phosphate, by Merck Group (2 mentions)
Erythrose 4 phosphate, by Merck Group (2 mentions)
Nah14co3, by PerkinElmer (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Sodium cyanoborohydride, by Merck Group (1 mentions)
4 protocols using ribulose 5 phosphate
1
Protein Purification and Kinetic Assay
2
Metabolite Labeling with AMC
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The metabolic extracts and sugar standards in methanol/water were labelled with 3-amino-9-methylcarbazole (AMC) prior to analysis. In detail, 50 µl of either a sugar mixture consisting of glucose-6 phosphate (Roche), mannose 6-phosphate (Sigma), ribose 5-phosphate (Sigma), ribulose 5-phosphate (Sigma), erythrose 4-phosphate (Sigma), and dihydroxyacetone phosphate (Sigma) or the metabolite extracts were mixed with 50 µl of 25 mM AMC (Enamine, Ukraine), 25 µl 50 mM sodium cyanoborohydride (Sigma), and 10 µl acetic acid. The reaction was kept at 70 °C for 60 min.
3
Hps–phi Enzyme Activity Assay
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The fused enzyme activity of purified Hps–phi was assayed by measuring the NADPH formation via coupled reactions catalyzed by glucose-6-phosphate dehydrogenase (G6PD) and phosphoglucoisomerase (Pgi) at 37 °C as described previously [33 (link),34 (link)]. Subsequently, 5 mM ribose-5-phosphate (Sigma-Aldrich, St. Louis, MO, USA) was used as the initial substrate to convert the reaction mixture of 200 µL to ribulose-5-phosphate by 10 U Pgi at 37 °C for 5 min to start the reaction. The reaction mixture contained 50 mM K2HPO4, 2.5 mM NADP+, 5 mM MgSO4, 10 U G6PD, and 10 U Pgi. To start the reaction, 5 mM formaldehyde was added to the reaction mixture. Each sample was tested in 3 parallels.
4
Phosphorylated Sugar Derivatization Protocol
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The phosphorylated sugars glucose-6-phosphate (Roche), mannose 6-phosphate (Sigma), ribose 5-phosphate (Sigma), ribulose 5-phosphate (Sigma), erythrose 4-phosphate (Sigma), and dihydroxyacetone phosphate (Sigma) in methanol/water were derivatized as described above for the metabolite extracts except that a deuterated version of AMC (3-amino-9-methyl-d3-carbazole (AMd3C), Enamine, Ukraine) was used for derivatization. The derivatized sugars were purified as described below and mixed to create a solution with a concentration of 80 fmol µl−1 for all sugar phosphates except ribulose 5-phosphate that was 800 fmol µl−1.
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