Open AccessThe large glycoprotein subunit of the skeletal muscle voltage‐sensitive calcium channel
Author(s) -
BURGESS Alison J.,
NORMAN Robert I.
Publication year - 1988
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1988.tb14479.x
Subject(s) - chemistry , g alpha subunit , skeletal muscle , dihydropyridine , glycosylation , protein subunit , biochemistry , dissociation constant , calcium channel , calcium , molecular mass , biophysics , enzyme , biology , receptor , endocrinology , organic chemistry , gene
Deglycosylation was used to assess the size of the core polypeptide of the large α 2 ‐glycoprotein subunit of the 1,4‐dihydropyridine‐sensitive calcium channel from rabbit skeletal muscle. The extent of glycosylation was assessed by measuring the shift in apparent molecular mass of the α 2 component following electrophoresis in sodium dodecyl sulphate/polyacrylamide gels, using anti‐(α‐subunit) monoclonal antibody staining of immunoblots. Chemical deglycosylation with trifluoromethanesulphonic acid produced a shift in apparent molecular mass of the α 2 component from M r 140 000 to M r 105 000, consistent with a carbohydrate content of approximately 25%. Enzymatic treatments were insufficient to deglycosylate the α 2 subunit fully, possibly due to the inaccessibility of glycosidic bonds to enzyme attack. Enzymatic deglycosylation procedures did, however, reduce the 1,4‐dihydropyridine‐binding activity of transverse‐tubule membranes. Neuraminidase alone or together with endo‐β‐ N ‐acetylglucosaminidase (endoglycosidase F) reduced the number of sites for (–)[ 3 H]PN 200–110 by 73 ± 2% and 77 ± 5% respectively, with no change in apparent dissociation constant, implying a possible role for the glycosylated subunits in the binding of 1,4‐dihydropyridines to the calcium‐channel complex. The development of the α 2 component in rat skeletal muscle was shown to be indistinguishable from the appearance of 1,4‐dihydropyridine binding activity consistent with the involvement of the α 2 subunit in the calcium‐channel complex at all stages of development.