Metabolically 35S-labeled recombinant calmodulin as a ligand for the detection of calmodulin-binding proteins

Judith Asselin, Sylvain Phaneuf, D. Martin Watterson, Jacques Haiech*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

We have developed a simplified procedure for the production of metabolically labeled calmodulin. We used bacterial clones (Escherichia coli) that were found to express VU-1 calmodulin, a calmodulin that is fully active with a variety of calmodulin-regulated enzymes. VU-1 calmodulin was labeled with sulfur-35 in bacteria maintained in a sulfur-free medium. Calmodulin was then purified by chromatography on phenyl-Sepharose. Under these conditions, the specific activity of the proteins was 150 to 400 cpm/fmol of calmodulin. To demonstrate the utility of this labeled VU-1 calmodulin, we examined the calmodulin-binding proteins in aortic myocyte preparation from Day 0 and Day 15 cultures by using both the gel and the nitrocellulose overlay protocols. The results showed that calmodulin-binding proteins are easily detected by the two procedures and that the profile of these target proteins changed in myocyte with time in culture. While most of these calmodulin-binding proteins have not been identified, the relative mobility on SDS-PAGE gels suggests that myosin light chain kinase (Mr ≈ 137,000) was detected by these methods. We demonstrated here that the nitrocellulose overlay was faster than the gel overlay and that this technique can be useful for the study of calmodulin-binding proteins.

Original languageEnglish (US)
Pages (from-to)141-147
Number of pages7
JournalAnalytical Biochemistry
Volume178
Issue number1
DOIs
StatePublished - Apr 1989

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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