Abstract
We have used subunit-specific antibodies to identify and to characterize partially the alpha, beta, gamma, and delta subunits of rat skeletal muscle acetylcholine receptor (AChR) on immunoblots. The alpha subunit of rat muscle is a single band of 42 kDa, whereas the beta subunit has an apparent molecular mass of 48 kDa. Both alpha and beta subunits are glycosylated and contain one or more N-linked oligosaccharide chains that are sensitive to endoglycosidase H digestion. The gamma and delta subunits, on the other hand, each appear as doublets on immunoblots, with apparent molecular masses of 52 kDa (gamma), 48 kDa (gamma') and 58 kDa (delta), 53 kDa (delta'), respectively. In each case, the two bands are structurally related and the lower band is probably the partial degradation product of the corresponding upper band. Each of the four gamma and delta polypeptides is N-glycosylated and contains both endoglycosidase H-sensitive and endoglycosidase H-resistant oligosaccharides. When the AChRs purified from embryonic, neonatal, adult, and denervated adult rat muscles were compared, no differences in the mobilities of alpha, beta, or delta subunits on sodium dodecyl sulfate gels were detected among them, either with or without endoglycosidase treatment. The gamma subunits, which were present in AChRs purified from neonatal, embryonic, or denervated rat muscles, were also identical; no gamma subunit was detected, however, in AChRs of normal adult rat muscle.
Highlights
From the Division of Neurobiology, Department of Physiology, University of California, San Francisco, San Francisco, California 94143-0444
To investigate further themolecularbasis of changes during development, we have prepared specific antibodies against synthetic peptides with amino acid sequences that are unique to the y and 6 subunits, respectively, of the mousemuscle AChR [29].We report here the use of these antibodies, along with subunit-specific monoclonal antibodies, to identify and to compare on protein blots each of the four subunits (a,p, The nicotinic acetylcholine receptor (AChR)l is a transmembrane glycoprotein that binds the neurotransmitter acetylcholine (ACh) and mediates synaptic transmission at the neuromuscular junction and artelated synapses in theelectric y, and 6) of the AChR in developing and in denervated and normal adult rat muscle
The band migrated at an apparent molecular mass of 42 kDa, similar to thatof the a subunit of the Torpedo AChR (Fig. la, lane I). monoclonal antibodies (mAbs) 124, which recognizes the @ subunit of the Torpedo AChR and cross-reacts with the corresponding subunit of the mammalian muscle AChR [11, 13, 30], identified a single band from the receptor preparation, in this case with an apparent molecular mass of 48kDa(Fig. IC).When immunoblots of control receptor preparations were tested, no bands were labeled by any of the three antibodies (Fig. 1, a, b, and c, lanes 3), demonstrating that the bands recognized bymAbs 14-3-F7 and 61 or by mAb 124 were associated with the receptor
Summary
Ments, the extract(S2) was split into two parts. One part was processed as described above to isolate AChRs. Identification of Mammalian Muscle AChR Subunits essed identically but was incubated with excess a-BuTx for 2 hbefore application to theaffinity column. This sample was used as a control for nonspecific binding of proteins to theaffinity column. The reaction mixture was applied to a Bio-Gel P2 column and eluted with 10 mM Na/PO,, pH 7.4, to separate the derivatized toxin from unreacted ANB-A1. The proteins in the gel were electrophoretically transferred t o nitrocellulose membranes and probed with antibodies specific for a,p, y,or 6 subunits as described below. The a and Subunits-The a and /3 subunits of mammalian incubated with about 10 nMof derivatized a-BuTx with or without 100-fold excess of underivatized toxin for 2 h a t 37 "C in the dark. The samples were mixed with equal of affinity-purified AChRs with specific antibodies
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