STRUCTURAL AND LIGAND RECOGNITION PROPERTIES OF IMIDAZOLINE BINDING-PROTEINS IN TISSUES OF RAT AND RABBIT

Imidazoline/guanidinium receptive sites (IGRS) belong to a family of membrane proteins that selectively recognize certain pharmacologically active compounds with an imidazoline or a guanidinium moiety. The role of such proteins in the cellular responses elicited by these compounds is unclear, but two members of this protein family are identical to isoforms of monoamine oxidase, an enzyme involved in the metabolism of monoamine neurotransmitters. To characterize the structural and ligand recognition properties of the imidazoline binding proteins, we used the photoaffinity adduct [I-125]iodoazidophenoxymethylimidazoline ([I-125]AZIPI) to label their ligand binding subunits in selected target tissues (kidney, pancreatic B cells, liver, and salivary gland). Photoaffinity labeling of membrane preparations or subcellular particulate fractions from various rat, rabbit, or hamster tissues indicated two labeled peptides of M(r) similar to 55,000 and similar to 61,000, the relative tissue distribution of which mirrored the expression of the A or B isoforms of monoamine oxidase. The ligand binding subunit of imidazoline binding proteins was identified on two peptides of M(r) similar to 55,000 and similar to 61,000 in rat and rabbit kidney, rat liver, rabbit salivary gland, and the pancreatic B cell line RIN-5AH, whereas only an M(r) similar to 61,000 peptide was observed in rat salivary gland and the hamster pancreatic B cell line HIT-T15. Saturation labeling experiments indicated that [I-125]AZIPI exhibited similar affinity (K-d similar to 2-3 nM) for both the M(r) similar to 55,000 and similar to 61,000 peptides. However, competitive inhibition of photolabeling indicated that the two peptides were distinguished by their affinity for the guanidinium guanabenz or their interaction with potassium. Although some types of imidazoline binding sites are located on the enzyme monoamine oxidase, the nonisoform selective enzyme inhibitor pargyline did not alter photoaffinity labeling of either the M(r) similar to 55,000 or similar to 61,000 peptide, indicating that imidazolines/guanidiniums and active site inhibitors of monoamine oxidase interact with different domains on the enzyme. In rat kidney and liver, an additional photolabeled peptide of M(r) similar to 25,000 was observed, and its ligand recognition profile was distinct from the M(r) similar to 55,000 and similar to 61,000 species. In contrast with the mitochondrial location of the larger peptides, subcellular fractionation of liver homogenates indicated that the M(r) similar to 25,000 localized to the plasma membrane.