Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1980 Feb 11;8(3):623–633. doi: 10.1093/nar/8.3.623

Physical and kinetic properties of the site specific endonuclease Bam HI from Bacillus amylolique-faciens.

B Hinsch, M R Kula
PMCID: PMC327295  PMID: 6255448

Abstract

The site specific endonuclease Bam HI which is composed of subunits of a molecular weight of 22 000 [1] can aggregate to complexes of a molecular weight of 360 000. It is an acidic protein with an isoelectric point at pH 5.3. Optimal activity is reached at 13 mM MgCl2. A very simple method is presented to determine kinetic constants of restriction enzymes directly from agarose gel photographs without any further equipment applying the integrated Michaelis Menten equation. With pJC 80 DNA as a substrate KM was found to be 3.6 10(-10) M. The method can be used to redefine the unit activity of site specific endonucleases unambigously.

Full text

PDF
623

Selected References

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

  1. Berg O. G., Blomberg C. Association kinetics with coupled diffusion. An extension to coiled-chain macromolecules applied to the lac repressor-operator system. Biophys Chem. 1977 Jun;7(1):33–39. doi: 10.1016/0301-4622(77)87012-9. [DOI] [PubMed] [Google Scholar]
  2. Catterall J. F., Welker N. E. Isolation and properties of a thermostable restriction endonuclease (ENDO R-Bst1503). J Bacteriol. 1977 Feb;129(2):1110–1120. doi: 10.1128/jb.129.2.1110-1120.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Goebel W., Bonewald R. Class of small multicopy plasmids originating from the mutant antibiotic resistance factor R1 drd-19B2. J Bacteriol. 1975 Aug;123(2):658–665. doi: 10.1128/jb.123.2.658-665.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  6. Modrich P., Zabel D. EcoRI endonuclease. Physical and catalytic properties of the homogenous enzyme. J Biol Chem. 1976 Oct 10;251(19):5866–5874. [PubMed] [Google Scholar]
  7. Roberts R. J. Restriction and modification enzymes and their recognition sequences. Gene. 1978 Nov;4(3):183–194. doi: 10.1016/0378-1119(78)90017-3. [DOI] [PubMed] [Google Scholar]
  8. Roberts R. J. Restriction endonucleases: a new role in vivo? Nature. 1978 Feb 9;271(5645):502–502. doi: 10.1038/271502a0. [DOI] [PubMed] [Google Scholar]
  9. Roberts R. J., Wilson G. A., Young F. E. Recognition sequence of specific endonuclease BamH.I from Bacillus amyloliquefaciens H. Nature. 1977 Jan 6;265(5589):82–84. doi: 10.1038/265082a0. [DOI] [PubMed] [Google Scholar]
  10. Rubin R. A., Modrich P. Substrate dependence of the mechanism of EcoRI endonuclease. Nucleic Acids Res. 1978 Aug;5(8):2991–2997. doi: 10.1093/nar/5.8.2991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  12. Smith L. A., Chirikjian J. G. Purification and characterization of the sequence-specific endonuclease Bam HI. J Biol Chem. 1979 Feb 25;254(4):1003–1006. [PubMed] [Google Scholar]
  13. Thomas M., Davis R. W. Studies on the cleavage of bacteriophage lambda DNA with EcoRI Restriction endonuclease. J Mol Biol. 1975 Jan 25;91(3):315–328. doi: 10.1016/0022-2836(75)90383-6. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES