Crystalline ribulose bisphosphate carboxylase/oxygenase of high integrity and catalytic activity from Nicotiana tabacum.
Crystalline tobacco (Nicotiana tabacum L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) was prepared using a procedure which protected the enzyme from hydrolysis by endogenous proteases. Leaves were extracted in a buffered medium containing casein, leupeptin, and high concentrations of MgSO4 and NaHCO3. After filtration through ion-exchange resin to remove contaminants, the enzyme was concentrated by precipitation with polyethylene glycol and crystal formation was induced by low-salt dialysis. The crystalline enzyme had a measured specific activity of 1.7 mumol CO2 mg protein-1 min-1, and about 93% of the enzyme could be activated with Mg2+ and CO2. Crystalline enzyme prepared in the absence of casein exhibited an activity which was only one-third of this rate and only about 70% of the enzyme could be activated with Mg2+ and CO2. Casein-extracted enzyme was resolved into distinct bands corresponding to the large (55,000) and small (14,000) subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit of enzyme prepared according to the latter procedure was found to be composed of five different polypeptides of slightly decreasing molecular weight. Only about one-third of the large subunits were of the 55,000 molecular weight type. No differences between the two preparations were observed in the Km (CO2) and apparent Km (ribulose bisphosphate).
D002384:Catalysis; D002498:Centrifugation; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010105:Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D010947:Plants, Toxic; D012273:Ribulose-Bisphosphate Carboxylase / Q000302:isolation & purification* / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology*
D009097:Multienzyme Complexes; D010105:Oxygenases; D012273:Ribulose-Bisphosphate Carboxylase