Subunit dissociation and reconstitution of ribulose-1,5-bisphosphate carboxylase from Chromatium vinosum.
The large and small subunits of ribulose bisphosphate carboxylase from Chromatium vinosum were dissociated and separated at pH 9.6 by sucrose density gradient centrifugation. After further purification by gel filtration, the small subunit fraction contained no carboxylase activity. The large subunit fraction was highly depleted of small subunit based on analysis by denaturing polyacrylamide gel electrophoresis. Carboxylase activity of the large subunit fraction was approximately 1% of the untreated native enzyme. Addition of purified small subunit to the large subunit fraction yielded increases of up to 67-fold in carboxylase activity, further indicating that both subunit types are required for catalysis by this enzyme. The isolated large subunit was fully capable of high-affinity activator 14CO2 binding in the presence of Mg2+ and 2-carboxyarabinitol bisphosphate, indicating that the activator and catalytic sites were not grossly denatured by the depletion of small subunit. Kinetic constants of the native C. vinosum enzyme defined a new class of ribulose bisphosphate carboxylase, which permits the detection of possible kinetic differences if the large and small subunits can be favorably reassembled with those of another kinetic class. From experiments with the enzymes from tobacco and spinach leaves it is concluded that the enzyme from higher plant sources is not suitable for such dissociation/reconstitution-type experiments.
D001665:Binding Sites; D002245:Carbon Dioxide; D002384:Catalysis; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D002844:Chromatium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium; D010428:Pentosephosphates; D010446:Peptide Fragments / Q000302:isolation & purification; D010944:Plants / Q000201:enzymology; D010947:Plants, Toxic; D012273:Ribulose-Bisphosphate Carboxylase / Q000302:isolation & purification*; D013045:Species Specificity; D013402:Sugar Alcohols; D014026:Nicotiana / Q000201:enzymology
D010428:Pentosephosphates; D010446:Peptide Fragments; D013402:Sugar Alcohols; D002245:Carbon Dioxide; C032687:2-carboxyarabinitol 1,5-biphosphate; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium