Calmodulin-binding proteins of the microfilaments present in isolated brush borders and microvilli of intestinal epithelial cells

Isolated microfilament cores of intestinal microvilli are known to contain actin and four major associated proteins among which is calmodulin. Immunofluorescence microscopy reveals that calmodulin is present in the microvilli prior to biochemical fractionation of intestinal cells and thus is not bou...

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Bibliographic Details
Published in:The Journal of biological chemistry 1980-11, Vol.255 (22), p.10551-10554
Main Authors: J R Glenney, Jr, K Weber
Format: Article
Language:English
Subjects:
Animals
Calcium-Binding Proteins - analysis
Calmodulin - analysis
Cell Membrane - analysis
Chickens
Intestinal Mucosa - analysis
Iodine Radioisotopes
Microvilli - analysis
Molecular Weight
Peptides - analysis
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Summary:Isolated microfilament cores of intestinal microvilli are known to contain actin and four major associated proteins among which is calmodulin. Immunofluorescence microscopy reveals that calmodulin is present in the microvilli prior to biochemical fractionation of intestinal cells and thus is not bound artifactually during the isolation procedure. Identification of the major microvillus calmodulin-binding protein was achieved by the use of an [125I]calmodulin gel overlay technique. Proteins of microvilli or brush borders were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After removal of sodium dodecyl sulfate, direct binding of radiolabeled calmodulin to the separated polypeptides was assayed by autoradiography. Three calmodulin-binding polypeptides are detected in brush borders. Two polypeptides (apparent Mr = 280,000 and 140,000) show Ca2+ -dependent binding, whereas the third polypeptide (Mr = 110,000) can bind calmodulin in the presence or absence of Ca2+. Microvillus core filaments contain only the latter species. Microvillus cores treated with 25 mM Mg2+ retain calmodulin and the 110,000 polypeptide, whereas the other two associated proteins are greatly reduced, consistent with the hypothesis that the 110,000 protein is the major calmodulin-binding protein of the core filament structure. We discuss the currently documentable structure of the core filaments and evaluate the general usefulness of the calmodulin gel overlay technique.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)70336-6