Molecular weight and structural nonequivalence of the mature alpha subunits of Torpedo californica acetylcholine receptor.

Abstract

A discrepancy of about 20% exists between the molecular weight of the a subunit of Torpedo californica electroplax acetylcholine receptor as determined by gel electrophoresis of the mature protein (Mr 40,000 ± 2000) and by nucleotide sequence analysis of cDNA (Mr z50,000 Each a subunit contains a high-affinity binding site for cholinergic ligands and possibly for a-bungarotoxin (1). These two sites are not equivalent. After reduction of a disulfide bridge close to the binding sites, they both can be covalently labeled by cholinergic agents such as bromoacetylcholine (9, 10) and one of them is more susceptible to such labeling (9), so that for many years it was believed that only one site on each AcChoR molecule could be labeled (11). The nonequivalence of the two sites can be explained by their different microenvironments since each a chain must be flanked by different subunits. However, the two a subunits themselves could be biochemically different, in spite of the identical primary structure of their precursors (3,4,8) and of the mature proteins (2). Another open question regards the exact molecular weight of the mature a subunit. The sequences deduced from its precursors consistently yielded a Mr of -50,000 both for Torpedo californica (4) and Torpedo marmorata AcChoR (8). This predicted value is considerably larger than the apparent molecular weight of the a subunit calculated from its behavior upon NaDodSO4 gel electrophoresis, which is between Mr 38,000 and Mr 44,000 depending on the gel system used (1). The possibility of a post-translational cleavage of a carboxyl-terminal peptide has been suggested (4), supported by the existence in this region of paired basic residues, which generally represent sites of proteolytic processing (12). To answer these questions, we have directly investigated the amino acid sequence of critical segments of the mature a subunits of T.californica AcChoR. MATERIALS AND METHODSPurification of the a Subunit. Membrane-bound AcChoR was isolated from T. californica electric organ by subcellular fractionation (13) followed by pH 11 extraction (13,14). The membrane fragments (25-50 mg of protein) were incubated in 1.5% NaDodSO4/5% glycerol/2.5% mercaptoethanol for 2 min at 90°C to achieve complete dissociation of the subunit. The sample was made 0.02% in bromophenol blue/2% in sodium thioglycolate and loaded on a preparative slab gel, according to Laemmli (15), containing 8.75% polyacrylamide. The running gel was 0.5 cm thick, 13 cm long, and 26 cm wide; the spacer gel was 1.5 cm long. The gels were run for -20 hr at 60 mA, stained with Coomassie blue for 6 hr, and destained for 24 hr as described (16). The stained protein bands were cut and stored frozen. The AcChoR subunits were recovered by electroelution and were electrodesalted (16). The NaDodSO4 used in the buffers for electroelution and desalting had been recrystallized twice from hot ethanol. The purity and the integrity of the isolated subunit were checked by NaDodSO4/polyacrylamide gel electrophoresis.The protein b...

Determination of the primary amino acid sequence specifying the alpha-bungarotoxin binding site on the alpha subunit of the acetylcholine receptor from Torpedo californica.

“…Our results agree with the findings of others who observe a 19-kDa MBTA-labeled fragment and a 17-kDa carbohydrate-containing fragment following digestion of the a subunit with V8 protease (15). It has been proposed by Conti-Tronconi et al (14) that the NH2 termini of both the 19-and 17-kDa fragments begin at AA 46 and that both fragments contain asparagine-141. Similarly, Lindstrom et al (16) propose that these two fragments begin at AA 42.…”

“…To use asparagine-141 as a reference point to map the BTX-binding site, it was necessary to demonstrate that all of the a subunit in the AcChoR possessed a marker for asparagine-141, namely an N-linked high-mannose oligosaccharide chain. This was especially important in light ofa recent report that the two a subunits in the AcChoR may differ in extent of glycosylation (14). Fig.…”

Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.

“…Conti-Tronconi et al also reported on another, larger fragment (19 kDa) that stains for carbohydrate only after prolonged staining with periodate/Schiff reagent and claimed that it is similar to their 17-kDa fragment in sequence, yet differs in the degree of glycosylation. If this were the case, then the 19-kDa fragment described by these authors (32) would not correspond to our 18-kDa fragment.…”

Activation of acetylcholine receptor channels by covalently bound agonists in cultured rat myoballs.

“…Extrasynaptic receptors display an antigenic site that synaptic receptors lack; it was suggested that this epitope might be a glyscosyl side chain on the receptor (Hall, Roisin, Gu & Gorin, 1983). Furthermore, the two a-subunits of the receptor seem to be differentially glyscosylated (Conti-Tronconi, Hunkapiller & Raftery, 1984). It is not yet known whether these differences in glycosyl side chains underlie the differential alkylation of the a-subunits by the tethered agonists and antagonists.…”