Purification and characterization of dihydropyridine receptor from rabbit skeletal muscle.
Digitonin and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate (Chapso) were used to solubilize the receptor of dihydropyridine calcium antagonists from the transverse tubule membranes of rabbit skeletal muscle. The receptor retained the ability for selective adsorption from either detergent extract by dihydropyridine-Sepharose. Incubation of the affinity resin with nitrendipine resulted in the elution of the receptor protein composed of two main polypeptides with molecular masses of 160 kDa and 53 kDa, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only these two subunits were found in the receptor preparation purified to a specific dihydropyridine-binding activity of 2500-2800 pmol/mg protein (60-70% purity) from digitonin-solubilized membranes by a combination of wheat-germ-agglutinin--Sepharose, anion-exchange and dihydropyridine-Sepharose chromatography steps. The individual subunits were isolated in dodecyl-sulfate-denatured form from the preparation of the receptor, enriched by a two-step large-scale procedure applied to Chapso-solubilized membranes. The 160-kDa subunit slowly changed its apparent molecular mass to 125 kDa upon disulfide bond reduction without formation of novel peptides. This finding implies that 160-kDa subunit is cross-linked by intramolecular S-S bridge(s). Chemical deglycosylation with trifluoromethanesulfonic acid showed that the carbohydrate content of large and small subunits accounted for 7.5% and 6.6% by mass, respectively. The dihydropyridine receptor subunits are glycosylated through N-glycoside bonds only. In their ratio of polar to hydrophobic amino acid residues in the amino acid composition of the receptor subunits, these polypeptides behave rather as peripheral proteins. It is suggested that the main portion of polypeptide chains is located outside the membrane in contact with solvent.
Soldatov
N M
NM
Institute of Biomedical Technology, USSR Ministry of Public Health, Moscow, USSR.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015220:Calcium Channels; D002793:Cholic Acids / Q000494:pharmacology; D002846:Chromatography, Affinity; D004072:Digitonin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D009132:Muscles / Q000032:analysis*; D010455:Peptides / Q000302:isolation & purification; D011485:Protein Binding; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000302:isolation & purification*; D012995:Solubility
D000596:Amino Acids; D015220:Calcium Channels; D002793:Cholic Acids; D010455:Peptides; D011978:Receptors, Nicotinic; C048531:chapso; D004072:Digitonin