Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1970 Dec;104(3):1065–1073. doi: 10.1128/jb.104.3.1065-1073.1970

Paraffin Oxidation in Pseudomonas aeruginosa II. Gross Fractionation of the Enzyme System into Soluble and Particulate Components

J Van Eyk 1, T J Bartels 1
PMCID: PMC248262  PMID: 16559078

Abstract

Osmoplast production in Pseudomonas aeruginosa was investigated to obtain osmotically sensitive cells for studies on the subcellular location of the paraffin-oxidizing enzyme system. It proved possible to convert cells of P. aeruginosa treated with lysozyme and ethylenediaminetetraacetic acid in tris(hydroxymethyl)aminomethane-sucrose buffer (pH 8) into osmotically sensitive cells within 2 min. Active, cell-free preparations were obtained by the subsequent osmotic disruption in the presence of deoxyribonuclease and magnesium chloride. The conditions necessary for a complete separation of membranes and soluble cell constituents were established by following the distribution of two reference enzymes. An enzyme assay based on direct gas chromatographic analysis of the oxidation products from n-heptane is described for the paraffin-oxidizing enzyme system. By using this method, we investigated the enzymatic organization and subcellular distribution of the paraffin-oxidizing enzyme system. It was confirmed that the enzyme system is composed of three components, each of which is indispensable for the hydroxylation of n-heptane. One of these components, the hydroxylase, was located in two cell fractions; the other two components occur exclusively in the soluble cell fraction. The half-life of a crude enzyme preparation kept at ambient temperature is approximately 3.5 hr. This poor stability was found to be primarily due to the instability of one of the soluble factors, presumably the reduced nicotinamide adenine dinucleotide-rubredoxin reductase.

Full text

PDF
1065

Selected References

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

  1. Asbell M. A., Eagon R. G. Role of Multivalent Cations in the Organization, Structure, and Assembly of the Cell Wall of Pseudomonas aeruginosa. J Bacteriol. 1966 Aug;92(2):380–387. doi: 10.1128/jb.92.2.380-387.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birdsell D. C., Cota-Robles E. H. Production and ultrastructure of lysozyme and ethylenediaminetetraacetate-lysozyme spheroplasts of Escherichia coli. J Bacteriol. 1967 Jan;93(1):427–437. doi: 10.1128/jb.93.1.427-437.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. CAMPBELL J. J., HOGGLA, STRASDINE G. A. Enzyme distribution in Pseudomonas aeruginosa. J Bacteriol. 1962 May;83:1155–1160. doi: 10.1128/jb.83.5.1155-1160.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cardini G., Jurtshuk P. Cytochrome P-450 involvement in the oxidation of n-octane b cell-free extracts of Corynebacterium sp. strain 7E1C. J Biol Chem. 1968 Nov 25;243(22):6070–6072. [PubMed] [Google Scholar]
  5. DIENA B. B., WALLACE R., GREENBERG L. THE PRODUCTION AND PROPERTIES OF SALMONELLA TYPHI SPHEROPLASTS. Can J Microbiol. 1964 Aug;10:543–549. doi: 10.1139/m64-068. [DOI] [PubMed] [Google Scholar]
  6. DIENES L., ZAMECNIK P. C. Transformation of bacteria into L forms by amino acids. J Bacteriol. 1952 Nov;64(5):770–771. doi: 10.1128/jb.64.5.770-771.1952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eagon R. G., Asbell M. A. Disruption of activity of induced permeases by tris(hydroxymethyl)amino methane in combination with ethylenediaminetetraacetate. Biochem Biophys Res Commun. 1966 Jul 6;24(1):67–73. doi: 10.1016/0006-291x(66)90411-6. [DOI] [PubMed] [Google Scholar]
  8. Heppel L. A. Selective release of enzymes from bacteria. Science. 1967 Jun 16;156(3781):1451–1455. doi: 10.1126/science.156.3781.1451. [DOI] [PubMed] [Google Scholar]
  9. Ichihara K., Kusunose E., Kusunose M. Microsomal hydroxylation of decane. Biochim Biophys Acta. 1969 Jun 10;176(4):713–719. [PubMed] [Google Scholar]
  10. Kusunose M., Matsumoto J., Ichihara K., Kusunose E., Nozaka J. Requirement of three proteins for hydrocarbon oxidation. J Biochem. 1967 May;61(5):665–667. doi: 10.1093/oxfordjournals.jbchem.a128600. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Lessie T., Neidhardt F. C. Adenosine triphosphate-linked control of Pseudomonas aeruginosa glucose-6-phosphate dehydrogenase. J Bacteriol. 1967 Apr;93(4):1337–1345. doi: 10.1128/jb.93.4.1337-1345.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Martin H. H. Bacterial protoplasts--a review. J Theor Biol. 1963 Jul;5(1):1–34. doi: 10.1016/0022-5193(63)90034-1. [DOI] [PubMed] [Google Scholar]
  14. Martin H. H. Biochemistry of bacterial cell walls. Annu Rev Biochem. 1966;35:457–484. doi: 10.1146/annurev.bi.35.070166.002325. [DOI] [PubMed] [Google Scholar]
  15. McKenna E. J., Kallio R. E. The biology of hydrocarbons. Annu Rev Microbiol. 1965;19:183–208. doi: 10.1146/annurev.mi.19.100165.001151. [DOI] [PubMed] [Google Scholar]
  16. Miyake Y., Gaylor J. L., Mason H. S. Properties of a submicrosomal particle containing P-450 and flavoprotein. J Biol Chem. 1968 Nov 10;243(21):5788–5797. [PubMed] [Google Scholar]
  17. Peterson J. A., Basu D., Coon M. J. Enzymatic omega-oxidation. I. Electon carriers in fatty acid and hydrocarbon hydroxylation. J Biol Chem. 1966 Nov 10;241(21):5162–5164. [PubMed] [Google Scholar]
  18. Peterson J. A., Coon M. J. Enzymatic omega-oxidation. 3. Purification and properties of rubredoxin, a component of the omega-hydroxylation system of Pseudomonas oleovorans. J Biol Chem. 1968 Jan 25;243(2):329–334. [PubMed] [Google Scholar]
  19. Peterson J. A., Kusunose M., Kusunose E., Coon M. J. Enzymatic omega-oxidation. II. Function of rubredoxin as the electron carrier in omega-hydroxylation. J Biol Chem. 1967 Oct 10;242(19):4334–4340. [PubMed] [Google Scholar]
  20. REPASKE R. Lysis of gram-negative bacteria by lysozyme. Biochim Biophys Acta. 1956 Oct;22(1):189–191. doi: 10.1016/0006-3002(56)90240-2. [DOI] [PubMed] [Google Scholar]
  21. Rothfield L., Finkelstein A. Membrane biochemistry. Annu Rev Biochem. 1968;37:463–496. doi: 10.1146/annurev.bi.37.070168.002335. [DOI] [PubMed] [Google Scholar]
  22. Salton M. R. Structure and function of bacterial cell membranes. Annu Rev Microbiol. 1967;21:417–442. doi: 10.1146/annurev.mi.21.100167.002221. [DOI] [PubMed] [Google Scholar]
  23. Siekevitz P. Electron transport systems in microsomes. Origin and functional nature of microsomes. Fed Proc. 1965 Sep-Oct;24(5):1153–1155. [PubMed] [Google Scholar]
  24. Simpson E. R., Estabrook R. W. Mitochondrial malic enzyme: the source of reduced nicotinamide adenine dinucleotide phosphate for steroid hydroxylation in bovine adrenal cortex mitochondria. Arch Biochem Biophys. 1969 Jan;129(1):384–395. doi: 10.1016/0003-9861(69)90190-8. [DOI] [PubMed] [Google Scholar]
  25. van Eyk J., Bartels T. J. Paraffin oxidation in Pseudomonas aeruginosa. I. Induction of paraffin oxidation. J Bacteriol. 1968 Sep;96(3):706–712. doi: 10.1128/jb.96.3.706-712.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. van der Linden A. C., Thijsse G. J. The mechanisms of microbial oxidations of petroleum hydrocarbons. Adv Enzymol Relat Areas Mol Biol. 1965;27:469–546. doi: 10.1002/9780470122723.ch10. [DOI] [PubMed] [Google Scholar]

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

RESOURCES