Skip to content
1887

Microbiology Society logo Microbiology

Volume 138, Issue 4

Multi-enzyme complexes in thermophilic organisms: isolation and characterization of the pyruvate dehydrogenase complex from AT62 Free

Abstract

Summary: The pyruvate dehydrogenase complex from the thermophilic bacterium was purified by Triton X-100 extraction and chromatography on phenyl-Sepharose CL-4B and HPLC-hydroxyapatite. The electrophoretic pattern of the purified enzyme complex was similar to that of the enzyme complex from , with four bands: the α-chain ( 39600) and β-chain ( 37500) of the pyruvate dehydrogenase component, the dihydrolipoamide acetytransferase component ( 58500) and the dihydrolipoamide dehydrogenase component ( 53900). Antibodies against the purified pyruvate dehydrogenase complex cross-reacted with the enzyme complex from and, to a minor extent, with that from bovine heart. No cross-reactivity could be observed with the enzyme complex from The enzyme complex had a temperature maximum at 72°C. 2-Oxobutyrate was a poor substrate and other 2-oxoacids were competitive inhibitors of the overall reaction. Long-chain 2-oxoacids showed a greater inhibitory effect, possibly caused by hydrophobic interactions. GTP inhibited the enzyme activity. Regulation of the pyruvate dehydrogenase complex from by allosteric mechanisms or by reversible phosphorylation could not be demonstrated.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Society for General Microbiology 1992
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-138-4-795
1992-04-01
2025-05-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/138/4/mic-138-4-795.html?itemId=/content/journal/micro/10.1099/00221287-138-4-795&mimeType=html&fmt=ahah

References

  1. Argos P., Rossmann M. G., Grau U. M., Zuber H., Frank G., Tratschin J. D. 1979; Thermal stability and protein structure. Biochemistry 18:5698–5703
     [Google Scholar]
  2. Barrera C. R., Namihara G., Hamilton L., Munk P., Eley M. H., Linn T. C., Reed L. J. 1972; α-Keto acid dehydrogenase complexes. XVI. Studies on the pyruvate dehydrogenase complexes from bovine kidney and heart. Archives of Biochemistry and Biophysics 148:343–358
     [Google Scholar]
  3. Bisswanger H. 1974; Regulatory properties of the pyruvate dehydrogenase complex from Escherichia coli. Thiamine pyrophosphate as an effector. European Journal of Biochemistry 48:377–387
     [Google Scholar]
  4. Bisswanger H. 1981; Substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli . Journal of Biological Chemistry 256:815–822
     [Google Scholar]
  5. Bisswanger H. 1984; Cooperativity in highly aggregated enzyme systems. A slow transition model for the pyruvate dehydrogenase complex from Escherichia coli . Journal of Biological Chemistry 259:2457–2466
     [Google Scholar]
  6. Blass J. P., Lewis C. A. 1973; Kinetic properties of the partially purified pyruvate dehydrogenase complex of ox brain. Biochemical Journal 131:31–37
     [Google Scholar]
  7. Bleile D. M., Hackert M. L., Pettit F. H., Reed L. J. 1981; Subunit structure of dihydrolipoyl transacetylase component of pyruvate dehydrogenase complex from bovine heart. Journal of Biological Chemistry 256:514–519
     [Google Scholar]
  8. Brock T. D., Freeze H. 1969; Thermus aquaticus gen.n. and sp.n., a nonsporulating extreme thermophile. Journal of Bacteriology 98:289–297
     [Google Scholar]
  9. Butler J. R., Pettit F. H., Davis P. F., Reed L. J. 1977; Binding of thiamin thiazolone pyrophosphate to mammalian pyruvate dehydrogenase and its effects on kinase and phosphatase activities. Biochemical and Biophysical Research Communications 74:1667–1674
     [Google Scholar]
  10. Degryse E., Glansdorff N., Piérard A. 1978; A comparative analysis of extreme thermophilic bacteria belonging to the genus Thermus . Archives of Microbiology 117:189–196
     [Google Scholar]
  11. DeMarcucci O., Lindsay J. G. 1985; Component X, an immunologically distinct polypeptide chain associated with mammalian pyruvate dehydrogenase multi-enzyme complex. European Journal of Biochemistry 147:641–648
     [Google Scholar]
  12. Dietrich J., Henning U. 1970; Regulation of the pyruvate dehydrogenase complex synthesis in Escherichia coli K12. Identification of the inducing metabolite. European Journal of Biochemistry 14:258–269
     [Google Scholar]
  13. Engel-Rae M., Henning U. 1973; Synthesis of the E. coli pyruvate dehydrogenase complex: non-dependence on 3′-5′-cyclic AMP. Biochemical and Biophysical Research Communications 53:812–817
     [Google Scholar]
  14. Fairbanks G., Steck T. L., Wallach T. F. H. 1971; Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606–2627
     [Google Scholar]
  15. Gebhardt C., Mecke D., Bisswanger H. 1978; Dihydrolipoamide transacetylase from Escherichia coli: evidence for internal gene duplication. Biochemical and Biophysical Research Communications 84:508–514
     [Google Scholar]
  16. Guest J. R. 1978; Aspects of the molecular biology of lipoamide dehydrogenase. Advances in Neurology 21:219–244
     [Google Scholar]
  17. Hartree E. F. 1972; Determination of protein: a modification of the Lowry method that gives linear photometric responses. Analytical Biochemistry 48:422–427
     [Google Scholar]
  18. Henderson C. E., Perham R. N., Finch J. T. 1979; Structure and symmetry of B. stearothermophilus pyruvate dehydrogenase complex and implications for eucaryote evolution. Cell 17:85–93
     [Google Scholar]
  19. Hensel R., Demharter W., Kandler O., Kroppenstedt R. M., Stackebrandt E. 1986; Chemotaxonomic and molecular-genetic studies of the genus Thermus: evidence for a phylogenetic relationship of Thermus aquaticus and Thermus ruber to the genus Deinococcus . International Journal of Systematic Bacteriology 36:444–453
     [Google Scholar]
  20. Heukeshoven J., Dernick R. 1985; Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6:103–112
     [Google Scholar]
  21. Jaenicke R. 1981; Enzymes under extremes of physical conditions. Annual Review of Biophysics and Bioengineering 10:1–67
     [Google Scholar]
  22. Junger E., Reinauer H., Wais U., Ullrich J. 1973; Electron microscopic study of pyruvate dehydrogenase from Saccharomyces carlsbergensis . Hoppe-Seyler's Zeitschrift für Physiologische Chemie 354:1655–1658
     [Google Scholar]
  23. Kresze G., -B. Ronft H. 1980; Bovine kidney pyruvate dehydrogenase complex. Limited proteolysis and molecular structure of the lipoate acetyltransferase component. European Journal of Biochemistry 112:589–599
     [Google Scholar]
  24. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
     [Google Scholar]
  25. Lehmacher A., Bisswanger H. 1988; Adaption of enzymes to high temperature. Isolation and characterization of the lipoamide dehydrogenase from Thermus aquaticus . Biochemistry (Life Science Advances) 7:29–33
     [Google Scholar]
  26. Martin R. G., Ames B. N. 1961; A method for determining the sedimentation behavior of enzymes: application to protein mixtures. Journal of Biological Chemistry 236:1372–1379
     [Google Scholar]
  27. Mellema J. E., van Bruggen E. S., Gruber M. 1967; Uranyl oxalate as a negative stain for electron microscopy of proteins. Biochimica et Biophysica Acta 140:180–182
     [Google Scholar]
  28. Morey A. V., Juni E. 1968; Studies on the nature of the binding of thiamine pyrophosphate to enzymes. Journal of Biological Chemistry 243:3009–3019
     [Google Scholar]
  29. Nickerson K. W. 1973; Biological functions of multistable proteins. Journal of Theoretical Biology 40:507–515
     [Google Scholar]
  30. Oshima T., Imahori K. 1974; Description of Thermus thermophilus comb.nov., a nonsporulating thermophilic bacterium from a Japanese thermal spa. International Journal of Systematic Bacteriology 24:102–112
     [Google Scholar]
  31. Packman L. C., Borges A., Perham R. N. 1988; Amino acid sequence analysis of the lipoyl and peripheral subunit-binding domains in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex from Bacillus stearothermophilus . Biochemical Journal 252:79–86
     [Google Scholar]
  32. Pettit F. H., Reed L. J. 1982; Pyruvate dehydrogenase complex from bovine kidney and heart. Methods in Enzymology 89:376–386
     [Google Scholar]
  33. Privalov P. L. 1979; Stability of proteins. Advances in Protein Chemistry 33:167–241
     [Google Scholar]
  34. Reed L. J. 1974; Multienzyme complexes. Accounts of Chemical Research 7:40–46
     [Google Scholar]
  35. Reed L. J. 1981; Regulation of mammalian pyruvate dehydrogenase complex by a phosphorylation–dephosphorylation cycle. Current Topics in Cellular Regulation 18:95–106
     [Google Scholar]
  36. Schmincke-Ott E., Bisswanger H. 1981; Dihydrolipoamide dehydrogenase component of the pyruvate dehydrogenase complex of Escherichia coli K12. Comparative characterization of the free and the complex-bound component. European Journal of Biochemistry 114:413–420
     [Google Scholar]
  37. Schwartz E. R., Reed L. J. 1970; Regulation of the activity of the pyruvate dehydrogenase complex of Escherichia coli . Biochemistry 9:1434–1439
     [Google Scholar]
  38. Teorell T., Stenhagen E. 1938; Ein Universalpuffer für den pH-Bereich 2,0−12,0. Biochemische Zeitschrift 299:416–419
     [Google Scholar]
  39. Visser J., Kester H., Huigen A. 1980; Purification and some properties of the pyruvate dehydrogenase complex of Bacillus subtilis . FEMS Microbiology Letters 9:227–232
     [Google Scholar]
  40. Weitzman P. D. J., Jaskowska-Hodges H. 1982; Patterns of nucleotide utilisation in bacterial succinate thiokinase. FEBS Letters 143:237–240
     [Google Scholar]
  41. Wieland O. H. 1983; The mammalian pyruvate dehydrogenase complex: structure and regulation. Reviews of Physiology and Biochemical Pharmacology 96:123–169
     [Google Scholar]
  42. Yeaman S. J. 1989; The 2-oxo acid dehydrogenase complexes: recent advances. Biochemical Journal 257:625–632
     [Google Scholar]
  43. Yoshida T., Lorence R. M., Choc M.-G., Tarr G. E., Findling K. L., Fee J. A. 1984; Respiratory proteins from the extremely thermophilic aerobic bacterium Thermus thermophilus . Journal of Biological Chemistry 259:112–123
     [Google Scholar]
  44. Zuber H. 1981; Structure and function of thermophilic enzymes. 32. Colloquium der Gesellschaft für Physiologische Chemie, pp. 114–127 Heidelberg: Springer-Verlag;
     [Google Scholar]
/content/journal/micro/10.1099/00221287-138-4-795
Loading
Multi-enzyme complexes in thermophilic organisms: isolation and characterization of the pyruvate dehydrogenase complex from Thermus aquaticus AT62
Microbiology 138, 795 (1992); https://doi.org/10.1099/00221287-138-4-795
/content/journal/micro/10.1099/00221287-138-4-795
/content/journal/micro/10.1099/00221287-138-4-795
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error