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Articles

Kinetic Variance of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Isolated from Diverse Taxonomic Sources ¹

II. Analysis by Two Dual Label Methods

Department of Microbiology and Biochemistry, University of Vermont, Burlington, Vermont 05405

Two dual label methods were used to investigate kinetic variability of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39). In addition to using [1-¹⁴C,5-³H]RuBP (method 1), we describe here the detailed assay with ¹⁴CO₂ and [5-³H]RuBP (method 2), which generates [³H,¹⁴C]3-phosphoglyceric acid and unlabeled (noncontaminating) phosphoglycolate; the carboxylase/oxygenase activity ratio (v_c/v_o) is calculated from ³H/¹⁴C ratios of substrates and products. v_c/v_o was found to be a linear function of [CO₂]/[O₂], constant over a 4-minute assay interval, and invariant of the degree of enzyme activity. Accurately measurable v_c/v_o ratios range from approximately 0.3 to 6. The K_m and V_max of both enzymes may be determined as a composite constant, V_cK_o/V_oK_c. By method 2, the directly compared, relative values at 40 micromolar CO₂ and 1240 micromolar O₂ were: Spinacia oleracea (74), Chlorella pyrenoidosa (31), Plectonema boryanum (32), and Rhodospirillum rubrum (8). With method 1, the values for S. oleracea and R. rubrum were 75, and 9, respectively. Under tight experimental controls, the absolute value for S. oleracea was 69 ± 3.