Stereoselective photoaffinity labelling of the purified 1,4-dihydropyridine receptor of the voltage-dependent calcium channel.
The voltage-dependent calcium channel from guinea-pig skeletal muscle T-tubules has been isolated with a rapid, two-step purification procedure. Reversible postlabelling of the channel-linked 1,4-dihydropyridine receptor and stereoselective photolabelling as a novel approach were employed to assess purity. A 135-fold purification to a specific activity of 1311 +/- 194 pmol/mg protein (determined by reversible equilibrium binding with (+)-[3H]PN200-110) was achieved. Three polypeptides of 155 kDa, 65 kDa and 32 kDa were identified in the purified preparation. The 155-kDa band is a glycoprotein. The arylazide photoaffinity probe (-)-[3H]azidopine bound with high affinity to solubilized membranes (Kd = 0.7 +/- 0.2 nM) and highly purified fractions (Kd = 3.1 +/- 2 nM), whereas the optical antipode (+)-azidopine was of much lower affinity. Irradiation of (-)-[3H]azidopine and (+)-[3H]azidopine receptor complexes with ultraviolet light led to preferential incorporation of the (-) enantiomer into the 155-kDa polypeptide in crude solubilized and purified preparations. The pharmacological profile of irreversible labelling of the 155-kDa glycoprotein by (-)-[3H]azidopine is identical to that found in reversible binding experiments. Specific photolabelling of the 155-kDa band by (-)-[3H]azidopine per milligram of protein increases 150-fold upon purification, whereas incorporation into non-specific bands in the crude solubilized material is identical for both, (-) and (+)-[3H]azidopine.
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D015220:Calcium Channels; D004095:Dihydropyridines; D006168:Guinea Pigs; D007473:Ion Channels / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D010777:Photochemistry; D011725:Pyridines; D011869:Radioligand Assay; D011978:Receptors, Nicotinic / Q000302:isolation & purification*; D012995:Solubility; D013237:Stereoisomerism
D000345:Affinity Labels; D001386:Azides; D015220:Calcium Channels; D004095:Dihydropyridines; D007473:Ion Channels; D011725:Pyridines; D011978:Receptors, Nicotinic; C041130:azidopine