The large glycoprotein subunit of the skeletal muscle voltage-sensitive calcium channel. Deglycosylation and development.
Deglycosylation was used to assess the size of the core polypeptide of the large alpha 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 alpha 2 component following electrophoresis in sodium dodecyl sulphate/polyacrylamide gels, using anti-(alpha 2-subunit) monoclonal antibody staining of immunoblots. Chemical deglycosylation with trifluoromethanesulphonic acid produced a shift in apparent molecular mass of the alpha 2 component from Mr 140,000 to Mr 105,000, consistent with a carbohydrate content of approximately 25%. Enzymatic treatments were insufficient to deglycosylate the alpha 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-beta-N-acetylglucosaminidase (endoglycosidase F) reduced the number of sites for (+)[3H]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 alpha 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 alpha 2 subunit in the calcium-channel complex at all stages of development.
Burgess
A J
AJ
Department of Medicine, Leicester Royal Infirmary, England.
Norman
R I
RI
D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D051381:Rats
D015220:Calcium Channels; D006023:Glycoproteins; D008565:Membrane Proteins; D010455:Peptides