Proteolytic nicking of the acetylcholine receptor.

Abstract

Low concentrations of papin rapidly cleave solubilized or membrane-bound acetylcholine receptor (AcChR) from Torpedo californica into a wide range of small fragments. The alpha subunits of the receptor are most resistant to cleavage. After solubilization in sodium dodecyl sulfate solutions the fragments are dissociated, and on electrophoresis the apparent subunit composition is reduced from four types (alpha, beta, gamma, and delta) to only alpha and finally, with large amounts of papain, to fragments even smaller than alpha. Prior to dissociation in sodium dodecyl sulfate, the proteolytic fragments remain physically and functionally associated. Thus, receptor which has been degraded so as to apparently contain only alpha subunits, or even no obvious subunits, still retains antigenic determinants corresponding to each subunit, still retains its characteristic size and doughnut shape when examined electron microscopically, and still sediments as dimers on sucrose gradients. Moreover, proteolytically nicked receptor remains fully functional in carbamylcholine-induced 22Na+ flux. These results demonstrate that inadequate inhibition of proteases during purification of receptor could account for reports from some laboratories that they have purified receptors containing only alpha subunits or fragments of alpha subunits. Also, our results demonstrate the strong noncovalent association between AcChR subunits which has thus far precluded their separation except under denaturing conditions in sodium dodecyl sulfate.