N-(3-PYRENE)MALEIMIDE - FLUORESCENT-PROBE OF ACETYLCHOLINE-RECEPTOR TRITON X-100 AGGREGATES

Acetylcholine receptor (AcChR) contains a disulfide bond which upon reduction can be labeled with a cholinergic analog, 4-(N-maleimide) α-benzyltrimethylammonium (MBTA) (Karlin, A. and Cowburn, D., 1973, Proc. Nat. Acad. Sci. USA 70, 3636-3660). A fluorescent reagent, N-(3-pyrene)maleimide, which reacts preferentially with protein thiols (Wu, C. W., Yarbrough, L. Z. and Hsiuch, Y., 1976, Biochemistry 15, 2863-2868), has been introduced into solubilized preparations of Torpedo californica AcChR. Both freshly isolated receptor and N-ethylmaleimide (NEM) (to protect exposed SH groups) and dithiothreitol (DTT) (to reduce the cholinergic site disulfide) treated receptor can be labeled with N-(3-pyrene)maleimide (PM). The attachment of the fluorescent probe to AcChR does not perturb the binding of α-bungarotoxin (α-Bgt) or of a cationic site ligand, propidium (Sator, V., Raftery, M. A., and Martinez-Carrion, M., 1977, Arch. Biochem. Biophys. 184, 95); it does, however, prevents labeling with MBTA. After treatment of AcChR with NEM and DTT, α-Bgt affords some protection against PM labeling. The PM probe inserted after reduction of the disulfide appears to rest on a cationic region(s) of the protein. Scrutiny of the fine vibrational structure, the total fluorescence or lifetime of the singlet excited state of bound PM show that binding of cholinergic ligands to AcChR does not alter the probe's environment. Fluorescence polarization parameters of bound PM gauge the extent of detergent receptor interaction in solubilized preparations. From the calculated correlation times a molar volume of 9.57 × 105 is obtained for AcChR Triton X-100 complexes which correspond to average Stokes' radii of 72Å. The latter values provide an explanation for the large discrepancy between the molecular weight of the receptor protein (270,000) and its anomalous behavior in gel filtration columns. © 1978.