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

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-(14)C,5-(3)H]RuBP (method 1), we describe here the detailed assay with (14)CO(2) and [5-(3)H]RuBP (method 2), which generates [(3)H,(14)C]3-phosphoglyceric acid and unlabeled (noncontaminating) phosphoglycolate; the carboxylase/oxygenase activity ratio (v(c)/v(o)) is calculated from (3)H/(14)C ratios of substrates and products. v(c)/v(o) was found to be a linear function of [CO(2)]/[O(2)], 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(c)K(o)/V(o)K(c). By method 2, the directly compared, relative values at 40 micromolar CO(2) and 1240 micromolar O(2) 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.