PURIFICATION OF RIBULOSE 1,5‐BISPHOSPHATE CARBOXYLASE AND CARBON ISOTOPE FRACTIONATION BY WHOLE CELLS AND CARBOXYLASE FROMCYLINDROTHECASP. (BACILLARIOPHYCEAE.)1,2,3

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Abstract
Ribulose 1,5‐biphosphate carboxylase has been purified to homogeneity from extracts ofCylindrothecasp. (strain N‐1), a marine, pennate diatom. The carboxylase has a molecular weight and structural composition similar to the enzyme from higher plants. When assayed in the presence of 1 mM NaHCO3the enzyme was stimulated nearly 40% by 1 mM aspartate and over 20% by 1 mM malate, and was inhibited to over 60% by 1 mM phosphoenolpyruvate. Similar experiments, using spinach carboxylase, failed to show activation by these metabolites. When assayed in the presence of 20 mM NaHCO3, 6‐phosphogluconate (1 mM) inhibited activity of ribulose bisphosphate carboxylase from Cylindrothecaby 60%, and higher concentrations of maiate (10 mM) inhibited activity by 25% Carbon isotope fractionation by ribulose bisphosphate carboxylase was ‐32.6% (ppt) when measured under N2using homogeneous enzyme, whereas maximum carbon isotope fractionation by the whole alga grown in 1% ‐C02‐in air averaged ‐ 16.8%. Carbon isotope fractionation by the whole alga varied with the density of the culture and was maximum at a low cell density (1.7 ± 106cellslml). At higher densities, the fractionation decreased by 4.0%. Carbon isotope fractionation has been used previously to determine the pathway of carbon metabolism in other organisms; the results of this investigation seem to indicate that this strain uses both the reductive pentose phosphate pathway and the C4carbon pathway for primary CO2fixation.