Solubilization and Characterization of Motilin-receptor Complexes from Rabbit Antral Smooth Muscle Tissue

Abstract

In the present study, the motilin receptor was characterized by enzymatic digestion studies and by solubilization of the motilin-receptor complex from prelabeled membranes using the anionic detergent cholic acid. Motilin binding was significantly decreased by preincubation of membranes of rabbit antral tissue with trypsin, phospholipase A₂, C, D, dithiothreitol and 2-mercaptoethanol but not by neuraminidase and β-galactosidase. Treatment of prelabeled membranes with 1% cholic acid resulted in solubilization of 24±5 % of the proteins and 65±3 % of the radioactivity. The latter was for 77±4 % due to the presence of the motilin-receptor complex as estimated with PEG-precipitation. Upon gelfiltration on Superose 6 the complex partially dissociated but 43±3 % eluted with macromolecular components in the void volume. This peak was not detected when membranes were first incubated with unlabeled motilin. Further disaggregation was accomplished by the addition of 0.5 M NaCl to the elution buffer. The chromatographic profile then showed a peak of about 370 kDa and a second one of 100kDa. The latter value probably reflects the molecular mass of a single ¹²⁵I-motilin-receptor-complex.