Purification and amino-terminal sequencing of the high affinity phenylalkylamine Ca2+ antagonist binding protein from guinea pig liver endoplasmic reticulum.

A high affinity phenylalkylamine Ca2+ antagonist binding polypeptide (Moebius, F. F., Burrows, G. G., Striessnig, J., and Glossmann, H. (1993) Mol. Pharmacol. 43, 139-148) was purified to homogeneity from the endoplasmic reticulum of guinea pig liver with the aid of [3H]emopamil, an antiischemic agent, and [3H]azidopamil, a photoaffinity label. The purified protein retained its high affinity for the antiischemic drugs emopamil (Kd = 4 nM), opipramol (IC50 = 15 nM), trifluoperazine (IC50 = 2 nM), and for Zn2+ (IC50 = 2 microM). Ferguson plots revealed a molecular mass of 27.2 kDa. Partial amino acid sequence information was obtained by Edman degradation and revealed no homology to known protein sequences. Antibodies raised against a synthetic peptide corresponding to the first 25 NH2-terminal amino acid residues specifically immunoprecipitated the [3H]azidopamil photoaffinity-labeled polypeptide and recognized the protein in Western blots. Cross-linking with a variety of homo- and heterobifunctional agents lead to the formation of dimers. Since in the purified preparation no other subunit could be identified with different protein stains, our results indicate that the [3H]emopamil binding site is formed by the homodimer of a novel membrane protein.