Effects of periodate oxidation and glycosidases on structural and functional properties of the acetylcholine receptor and the non-receptor, peripheral ?-polypeptide (M(r) 43,000).
NaIO(4) oxidation, exo- and endo-glycosidase treatments and combinations thereof have been applied to acetylcholine receptor from Torpedo marmorata in its membrane-bound and detergent-solubilised forms. The effects of these chemical and enzymatic treatments are made apparent in the electrophoretic properties of the four receptor subunits (?, ?, ? and ?) and of the non-receptor polypeptides, their thermal and proteolytic susceptibility, and the steady-state and kinetic parameters of receptor-toxin complex formation. The electrophoretic pattern of the membrane polypeptides is found to depend on the redox state of the membranes, presence or absence of the non-receptor peripheral ?-peptide (M(r) 43,000), pH and temperature. Very low NaIO(4) concentrations (50 ?M) suffice to prevent the penetration of the ?-peptide into NaDodSO(4) polyacrylamide gels. This effect could be abolished by N-ethylmaleimide alkylation of free sulphydryl groups, suggesting the involvement of easily oxidizable vicinal thiols in the aggregation of the peptide. Higher reagent concentrations resulted in the altered mobility and subsequent splitting of the receptor subunit carrying the ligand recognition site (?, M(r) 40,000) into a doublet. In contrast, NaIO(4) treatment of the detergent-solubilized receptor aggregated the ?-subunit, presumably via chemical groups hidden in the membrane but exposed in detergent. Only this subunit underwent such NaIO(4)-dependent changes within the concentration range in which (a) an increase of the 13-S dimeric receptor species at the expense of the 9-S monomeric form was observed and (b) half-maximal quenching of the intrinsic fluorescence occurred (?2 mM NaIO(4)). Neuraminidase digestion affected exclusively the ?- and ?-subunits of the receptor, suggesting the presence of substantial amounts of sialic acid residues in these subunits. ?-Glucosidase and endoglycosidase D had no effect on the electrophoretic properties of receptor and non-receptor polypeptides. Neither NaIO(4) nor neuroaminidase treatments had any effect on the thermal sensitivity of the receptor. Similarly, the equilibrium and kinetic properties of receptor-?-neurotoxin complex formation were not modified by such treatment nor was the susceptibility to tryptic digestion. The thermal and proteolytic sensitivities were affected by acid pH (5.2) and ?-glucosidase treatments. The latter enzymatic digestion reduced the ?-toxin binding capacity of the receptor by 35% and increased the equilibrium dissociation constant by 2-fold.
Criado
M
M
Max-Planck-Institut für biophysikalische Chemie, 3400 Göttingen, F.R.G.
Barrantes
F J
FJ