Ribulose diphosphate carboxylase from autotrophic microorganisms.
Thiobacillus denitrificans was grown anaerobically with nitrate as an acceptor in both sterile and nonsterile media. Ribulose diphosphate carboxylase was stable throughout the exponential growth phase and declined slowly only after cells reached the stationary phase. Reversible inactivation of the carboxylase occurred in extracts as a result of bicarbonate omission. The enzyme was purified 32-fold with excellent recovery of a preparation which was 50 to 60% pure by the criterion of polyacrylamide gel electrophoresis. This purified preparation catalyzed the fixation of 1.25 mumoles of CO(2) per min per mg of protein at pH 8.1 and 30 C, and the molecular weight of ribulose diphosphate carboxylase was approximately 350,000 daltons. A striking biphasic time course of CO(2) fixation that was independent of protein and ribulose diphosphate concentration was observed. The optimal pH of the enzyme assay was fairly broad, ranging from 7 to 8.2. Kinetic dependence upon bicarbonate, ribulose diphosphate, and Mg(2+) was characterized and indicated that bicarbonate and Mg(2+) must combine with enzyme prior to addition of ribulose diphosphate. Antiserum to ribulose diphosphate carboxylase from Hydrogenomonas eutropha was only slightly inhibitory when added to the enzyme from T. denitrificans, and the mixture did not precipitate. Cyanide (4 x 10(-5)m) gave 61% inhibition of the enzyme from T. denitrificans. Ribulose diphosphate carboxylase in extracts of H. eutropha, H. facilis, Chromatium D, Rhodospirillum rubrum, and Chlorella pyrenoidosa were also inhibited to varying extents by cyanide and antiserum to the H. eutropha enzyme.
D000693:Anaerobiosis; D001639:Bicarbonates; D002245:Carbon Dioxide / Q000378:metabolism; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D002474:Cell-Free System; D002708:Chlorella / Q000201:enzymology; D002844:Chromatium / Q000201:enzymology; D003470:Culture Media; D003486:Cyanides / Q000494:pharmacology; D004589:Electrophoresis, Disc; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D008274:Magnesium; D008970:Molecular Weight; D010428:Pentosephosphates; D011549:Pseudomonas / Q000201:enzymology; D012246:Rhodospirillum / Q000201:enzymology; D012247:Rhodospirillum rubrum / Q000201:enzymology; D013855:Thiobacillus / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D013997:Time Factors
D001639:Bicarbonates; D003470:Culture Media; D003486:Cyanides; D010428:Pentosephosphates; D002245:Carbon Dioxide; D002262:Carboxy-Lyases; D008274:Magnesium