Abstract
The 1,4-dihydropyridine receptor purified from rabbit skeletal muscle contains four polypeptide components of 175,000 Da (nonreduced)/150,000 Da (reduced), 170,000, 52,000, and 32,000 Da (Leung, A. T., Imagawa, T., and Campbell, K. P. (1987) J. Biol. Chem. 262, 7943-7946). A monoclonal antibody specific to the 52,000-Da polypeptide component of the dihydropyridine receptor has been produced and used in immunoprecipitation and immunoblotting experiments to demonstrate that the 52,000-Da polypeptide is an integral subunit of the purified dihydropyridine receptor. Peptide mapping experiments with 32P-labeled dihydropyridine receptor have also demonstrated that the 52,000-Da polypeptide is distinct from and not a proteolytic fragment of the 170,000-Da subunit. Densitometric scanning of Coomassie Blue-stained sodium dodecyl sulfate-polyacrylamide gels of the purified dihydropyridine receptor has demonstrated that the 52,000-Da polypeptide exists in a 1:1 stoichiometric ratio with the 170,000-, 175,000/150,000-, and 32,000-Da subunits of the dihydropyridine receptor. Electron microscopy of the freeze-dried, rotary-shadowed dihydropyridine receptor has shown that the preparation contains a homogeneous population of 16 x 22-nm ovoidal particles large enough to contain all four polypeptides of the dihydropyridine receptor. The particles have two distinct components of similar size which may represent the location in the molecule of the two larger subunits.
Highlights
From the $ Department of Physiology and Biophysics, The Universityof Iowa, Iowa City, Iowa 52242 and the Departments of
The immunodot assay positive hybridoma supernatants were tested on nitrocellulose transfers of SDS-polyacrylamide gels of partially purified dihydropyridine receptor and the void fraction of the WGASepharose column, which has been shown to be devoid of dihydropyridine receptor activity
The monoclonal antibody VD2, was found to staina 52,000-Da polypeptide in partially purified dihydropyridine receptor underboth nonreducing and reducing conditions but was not reactive against the void fraction of WGA-Sepharose (Fig. 1).This antibody was tested for its ability to immunoprecipitate the
Summary
Receptor-Ten pg of the purified dihydropyridine receptor from nine preparations, isolated using either digitonin or CHAPS for solubilization, were analyzed by SDS-PAGE (5-16% gradient gel) under both reducing and nonreducing conditions. 10-204 drop of solution containing 10-50 pg/ml of purified dihydropyridine receptor was applied to the surface of freshly cleaved mica. The mica was either gently rinsed with 10 drops of 50 mM Tris-HCI, pH 7.3, or directly exposed to 7-8 drops of 2% uranyl acetate containing 20 mg/ml bacitracin for 30 s and rinsed in 3% glycerol with bacitracin. Thisthin layer of solution was frozen by quick immersion of the mica in liquid nitrogen. Protease inhibitors and peroxidase-conjugated WGAwere obtained from Sigma. Digitonin was from Fisher and Sigma and prepared as described previously [7].All other reagents were of reagent grade quality
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