Immunological characterization of voltage-sensitive calcium cells.
thesisposted on 19.11.2015 by Alison J. Burgess
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A panel of monoclonal antibodies were raised against the 1,4-dihydropyridine sensitive Ca2+ channel of rabbit skeletal muscle. When tested on immunoblot assay of denatured and reduced transverse tubule membranes, four of the antibodies specifically recognized a polypeptide of Mr 140,000. This component co-migrated with the large glycoprotein ?2 subunit of purified Ca2+ channel preparations. On immunoblots of nonreducing gels the antibodies detected a component that migrated more slowly in the gel, with a Mr of 170,000, consistent with the disulphide-linkage of the ?2 subunit to a small component of Mr 30,000. Additionally, three of the antibodies also recognized high molecular weight components of Mr 310,000-330,000 under these conditions. Crossreactive polypeptides of similar apparent molecular weight were detected in immunoblot assays of rabbit heart and brain membranes and of skeletal muscle membranes from different species. Further similarities between the ?2 components of Ca2+ channels from different species were investigated by immunoblot assay, following the limited tryptic digestion of the skeletal muscle membranes. A similar pattern of immunoreactive peptides were detected in each case, suggesting that the ?2 subunits of Ca2+ channels from different species are similar, not only in terms of antibody binding sites but also with respect to similarly positioned trypsin cleavage sites. The extent of glycosylation of the ?2 component was investigated using enzymatic and chemical deglycosylation techniques. Chemical deglycosylation resulted in a core polypeptide of Mr 105,000, consistent with a carbohydrate content of approximately 25%. Enzymatic treatments, although insufficient to completely deglycosylate the ?2 component, reduced the maximal 1,4-dihydropyridine binding capacity of transverse tubule membranes by 73-77%. The co-development of the ?2 subunit with 1,4- dihydropyridine binding activity was shown in rat skeletal muscle. These results indicate that the '2 subunit is an integral structural component of the 1,4-dihydropyridine sensitive Ca2+ channel.