Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1972 May;110(2):633–642. doi: 10.1128/jb.110.2.633-642.1972

Ribulose Diphosphate Carboxylase from Autotrophic Microorganisms

Bruce A McFadden a, Anita R Denend a,1
PMCID: PMC247459  PMID: 4623310

Abstract

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 μmoles of CO2 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 CO2 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 Mg2+ was characterized and indicated that bicarbonate and Mg2+ 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 × 10−5m) 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.

Full text

PDF
633

Images in this article

637Image
on p.637

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akazawa T., Sato K., Sugiyama T. Structure and function of chloroplast proteins. 8. Some properties of ribulose-1,5-diphosphate carboxylase of athiorhodaceae in comparison with those of plant enzyme. Arch Biochem Biophys. 1969 Jun;132(1):255–261. doi: 10.1016/0003-9861(69)90360-9. [DOI] [PubMed] [Google Scholar]
  2. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  3. GOODWIN T. W., LAND D. G., OSMAN H. G. Studies in carotenogenesis. 14. Carotenoid synthesis in the photosynthetic bacterium Rhodopseudomonas spheroides. Biochem J. 1955 Mar;59(3):491–496. doi: 10.1042/bj0590491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. GOODWIN T. W., OSMAN H. G. Studies in carotenogenesis. 9. General cultural conditions controlling carotenoid (spirilloxanthin) synthesis in the photosynthetic bacterium Rhodospirillum rubrum. Biochem J. 1953 Mar;53(4):541–546. doi: 10.1042/bj0530541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kuehn G. D., McFadden B. A. Factors affecting the synthesis and degradation of ribulose-1,5-diphosphate carboxylase in Hydrogenomonas facilis and Hydrogenomonas eutropha. J Bacteriol. 1968 Mar;95(3):937–946. doi: 10.1128/jb.95.3.937-946.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kuehn G. D., McFadden B. A. Ribulose 1,5-diphosphate carboxylase from Hydrogenomonas eutropha and Hydrogenomonas facilis. I. Purification, metallic ion requirements, inhibition, and kinetic constants. Biochemistry. 1969 Jun;8(6):2394–2402. doi: 10.1021/bi00834a021. [DOI] [PubMed] [Google Scholar]
  7. Kuehn G. D., McFadden B. A. Ribulose 1,5-diphosphate carboxylase from Hydrogenomonas eutropha and Hydrogenomonas facilis. II. Molecular weight, subunits, composition, and sulfhydryl groups. Biochemistry. 1969 Jun;8(6):2403–2408. doi: 10.1021/bi00834a022. [DOI] [PubMed] [Google Scholar]
  8. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  9. PON N. G., RABIN B. R., CALVIN M. MECHANISM OF THE CARBOXYDISMUTASE REACTION. I. THE EFFECT OF PRELIMINARY INCUBATION OF SUBSTRATES, METAL ION AND ENZYME ON ACTIVITY. Biochem Z. 1963;338:7–19. [PubMed] [Google Scholar]
  10. RABIN B. R., TROWN P. W. MECHANISM OF ACTION OF CARBOXYDISMUTASE. Nature. 1964 Jun 27;202:1290–1293. doi: 10.1038/2021290a0. [DOI] [PubMed] [Google Scholar]
  11. REPASKE R. Nutritional requirements for Hydrogenomonas eutropha. J Bacteriol. 1962 Feb;83:418–422. doi: 10.1128/jb.83.2.418-422.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Spomer G. G. Molecular diversity of the ribulose-1,5-diphosphate carboxylase from photosynthetic microorganisms. Science. 1968 Aug 2;161(3840):482–485. [PubMed] [Google Scholar]
  13. Sugiyama T., Akazawa T. Ribulose-1,5-diphosphate carboxylase of Rhodopseudomonas spheroides. Biochem Biophys Res Commun. 1968 Jun 28;31(6):856–861. doi: 10.1016/0006-291x(68)90530-5. [DOI] [PubMed] [Google Scholar]
  14. Sugiyama T., Matsumoto C., Akazawa T., Miyachi S. Structure and function of chloroplast proteins. VII. Ribulose-1,5-diphosphate carboxylase of Chlorella ellipsoidea. Arch Biochem Biophys. 1969 Feb;129(2):597–602. doi: 10.1016/0003-9861(69)90219-7. [DOI] [PubMed] [Google Scholar]
  15. Sugiyama T., Nakayama N., Akazawa T. Structure and function of chloroplast proteins. V. Homotropic effect of bicarbonate in RuDP carboxylase reaction and the mechanism of activation by magnesium ions. Arch Biochem Biophys. 1968 Sep 10;126(3):737–745. doi: 10.1016/0003-9861(68)90465-7. [DOI] [PubMed] [Google Scholar]
  16. VISHNIAC W., SANTER M. The thiobacilli. Bacteriol Rev. 1957 Sep;21(3):195–213. doi: 10.1128/br.21.3.195-213.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. WOLIN E. A., WOLIN M. J., WOLFE R. S. FORMATION OF METHANE BY BACTERIAL EXTRACTS. J Biol Chem. 1963 Aug;238:2882–2886. [PubMed] [Google Scholar]
  18. Wishnick M., Lane M. D. Inhibition of ribulose diphosphate carboxylase by cyanide. Inactive ternary complex of enzyme, ribulose diphosphate, and cyanide. J Biol Chem. 1969 Jan 10;244(1):55–59. [PubMed] [Google Scholar]
  19. Wishnick M., Lane M. D., Scrutton M. C., Mildvan A. S. The presence of tightly bound copper in ribulose diphosphate carboxylase from spinach. J Biol Chem. 1969 Oct 25;244(20):5761–5763. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES