Interactions of the 18.5 kDa isoform of myelin basic protein with Ca2+-calmodulin: In vitro studies using gel shift assays

David S. Libich, George Harauz

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

The interactions of the 18.5 kDa isoform of myelin basic protein (MBP) with calmodulin (CaM) in vitro have been investigated using glutaraldehyde or dithiobis[succinimidylpropionate] (DSP) cross-linking, and SDS-polyacrylamide gel electrophoresis. The following forms of MBP were used: the natural bovine C1 charge isomer (bMBP/C1) and a recombinant murine product (rmMBP), and their fragments generated by digestion with cathepsin D (EC 3.4.23.5). In physiological buffers (10 mM HEPES-NaOH, pH 7.4,5 mM CaCl2, 0.0035% glutaraldehyde; or 50 mM HEPES-NaOH, pH 7.4, 100 mM NaCl, 1 mM CaCl2, 0.0035% DSP), MBP and CaM interacted primarily in a 1:1 molar ratio, consistent with previous studies that used 6 M urea, i.e. denaturing conditions. Moreover, the appearance of higher-order bands (not previously observed) suggested that the mechanism of interaction of the two proteins involved a series of relatively complex equilibria, resulting in 2:1 ratios of MBP to CaM. This observation would explain the cooperativity of association inferred from fluorescence studies [13]. Our results demonstrated further that the interaction involved the C-terminal domain of MBP, again in a primarily 1:1 molar ratio with CaM, consistent with our identification of a CaM-binding motif at the C-terminus.

Original languageEnglish (US)
Pages (from-to)45-52
Number of pages8
JournalMolecular and Cellular Biochemistry
Volume241
Issue number1-2
DOIs
StatePublished - Dec 1 2002
Externally publishedYes

Keywords

  • Ca-calmodulin
  • Cathepsin D
  • Myelin basic protein

ASJC Scopus subject areas

  • Molecular Biology
  • Clinical Biochemistry
  • Cell Biology

Fingerprint Dive into the research topics of 'Interactions of the 18.5 kDa isoform of myelin basic protein with Ca<sup>2+</sup>-calmodulin: In vitro studies using gel shift assays'. Together they form a unique fingerprint.

  • Cite this