Interactions of the 18.5 kDa isoform of myelin basic protein with Ca2+-calmodulin: in vitro studies using gel shift assays.
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.
Libich
David S
DS
Department of Molecular Biology and Genetics and Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada.
Harauz
George
G
D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005976:Glutaral / Q000737:chemistry; D066298:In Vitro Techniques; D004676:Myelin Basic Protein / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D004676:Myelin Basic Protein; D002402:Cathepsin D; D002118:Calcium; D005976:Glutaral