Ribulose 1,5-bisphosphate carboxylase from the halophilic cyanobacterium Aphanothece halophytica.
Various structural and functional properties of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isolated from the halophilic cyanobacterium (blue-green alga) Aphanothece halophytica were reexamined. The ready dissociation of this algal RuBisCO during sedimentation in a linear sucrose density gradient was observed. Low NaCl concentrations promote the dissociation of small subunit (B) from the original native enzyme molecule as evidenced by the sucrose density gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is thus possible that the intracellular osmoticum of A. halophytica might influence the structural integrity and activity of RuBisCO. The low residual carboxylase activity ascribed to the catalytic core, an oligomer form of the large subunit (A) apparently deficient in small subunit (B), was found to be markedly stimulated by a protein component which appears identical to subunit B. The purification and structural characterization of the catalytic core and subunit B were attempted by step-wise column chromatography on DEAE-cellulose, Utrogel AcA 34, Sephadex G-75, and hydroxylapatite, and at the final stage each component was purified to near homogeneity, although the catalytic core is still associated with a small quantity of subunit B. The addition of subunit B to the catalytic core does not alter the Km (HCO-3, RuBP) values, but Vmax values are markedly enhanced. Sucrose density gradient centrifugation gave a value of 16 S for the catalytic core. The molecular weights of the monomeric forms of the catalytic core (subunit A) and subunit B were 5.0 X 10(4) and 1.4 X 10(4), respectively.
D002499:Centrifugation, Density Gradient; D000458:Cyanobacteria / Q000201:enzymology*; D004789:Enzyme Activation; D005779:Immunodiffusion; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D012273:Ribulose-Bisphosphate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D012273:Ribulose-Bisphosphate Carboxylase