Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1985 Jun 1;228(2):375–381. doi: 10.1042/bj2280375

Monoclonal-antibody studies of creatine kinase. The proteinase K-cleavage site.

G E Morris, L C Frost, L P Head
PMCID: PMC1144995  PMID: 3893421

Abstract

Proteinase K cleaves a small peptide from native muscle-specific creatine kinase. We present evidence, from the binding of two monoclonal antibodies to formic acid-cleavage fragments and proteinase K-digest fragments of chick muscle creatine kinase, that the proteinase K-cleavage site is in the C-terminal region of the molecule. This specificity of proteinase K, which is not normally a highly specific enzyme, and the continued association of the two peptide fragments after cleavage suggest an unusual conformational feature in the cleavage-site region. By applying predictive methods for hydrophobicity and secondary structure to an amino acid sequence in this region, we suggest possible structural features at the cleavage site that are evidently conserved across avian and mammalian species. The most likely site is next to, or within, a beta-turn on the surface of the molecule.

Full text

PDF
375

Images in this article

376Fig. 1.
on p.376
377Fig. 2.
on p.377
378Fig. 3.
on p.378

Selected References

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

  1. Bowen B., Steinberg J., Laemmli U. K., Weintraub H. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 1980 Jan 11;8(1):1–20. doi: 10.1093/nar/8.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  3. Ebeling W., Hennrich N., Klockow M., Metz H., Orth H. D., Lang H. Proteinase K from Tritirachium album Limber. Eur J Biochem. 1974 Aug 15;47(1):91–97. doi: 10.1111/j.1432-1033.1974.tb03671.x. [DOI] [PubMed] [Google Scholar]
  4. Eppenberger H. M., Dawson D. M., Kaplan N. O. The comparative enzymology of creatine kinases. I. Isolation and characterization from chicken and rabbit tissues. J Biol Chem. 1967 Jan 25;242(2):204–209. [PubMed] [Google Scholar]
  5. Kwiatkowski R. W., Schweinfest C. W., Dottin R. P. Molecular cloning and the complete nucleotide sequence of the creatine kinase-M cDNA from chicken. Nucleic Acids Res. 1984 Sep 25;12(18):6925–6934. doi: 10.1093/nar/12.18.6925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  7. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  8. Morris G. E., Head L. P. A monoclonal antibody against the skeletal muscle enzyme, creatine kinase. FEBS Lett. 1982 Aug 16;145(1):163–168. doi: 10.1016/0014-5793(82)81228-3. [DOI] [PubMed] [Google Scholar]
  9. Morris G. E., Head L. P. Immunoassay of muscle-specific creatine kinase with a monoclonal antibody and application to myogenesis and muscular dystrophy. Biochem J. 1983 Aug 1;213(2):417–425. doi: 10.1042/bj2130417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Price N. C., Murray S., Milner-White E. J. The effect of limited proteolysis on rabbit muscle creatine kinase. Biochem J. 1981 Oct 1;199(1):239–244. doi: 10.1042/bj1990239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Price N. C., Stevens E. The refolding of denatured rabbit muscle creatine kinase. Search for intermediates in the refolding process and effect of modification at the reactive thiol group on refolding. Biochem J. 1982 Jan 1;201(1):171–177. doi: 10.1042/bj2010171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sonderegger P., Jaussi R., Gehring H., Brunschweiler K., Christen P. Peptide mapping of protein bands from polyacrylamide gel electrophoresis by chemical cleavage in gel pieces and re-electrophoresis. Anal Biochem. 1982 May 15;122(2):298–301. doi: 10.1016/0003-2697(82)90285-8. [DOI] [PubMed] [Google Scholar]
  13. Williamson J., Greene J., Chérif S., Milner-White E. J. Heterogeneity of rabbit muscle creatine kinase and limited proteolysis by proteinase K. Biochem J. 1977 Dec 1;167(3):731–737. doi: 10.1042/bj1670731. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES