The overall objectives of our laboratory are to determine the structural and biosynthetic relationships among the several forms of acetylcholinesterase (AChE) in mammalian tissues and the ways in which these forms are biologically regulated. The predominant form of AChE in neuromuscular junctions is an assembly of catalytic subunits and collagen-like noncatalytic subunits that appear to anchor this AChE form in the extracellular basement membrane. Alternative membrane-bound forms of AChE, found in nonjunctional muscle membrane, brain and erythrocytes, are devoid of collagen-like components and are localized directly in phospholipid membranes as integral membrane proteins. Human erythrocyte AChE (RBC AChE), which appears to be a good model for other integral membrane AChEs, is an amphipathic protein whose small hydrophobic membrane-binding domain is cleaved by certain proteases. A primary specific aim of this proposal is the amino acid sequence analysis of selected regions of the RBC AChE catalytic subunits. RBC AChE can be selectively radiolabeled at 4 sites: the hydrophobic domain can be labeled by a photolabeling reagent; the N-terminal amino acid, by reductive methylation; the single sulfhydryl involved in intersubunit disulfide bonding, by selective reduction and alkylation; and the active site, by organophosphorylation. RBC AChE peptides generated by CNBr and trypsin will be fractionated by gel exclusion chromatography and HPLC. Isolated peptides, particularly those selectively labeled, will be sequenced. Sequence information about the hydrophobic domain should clarify how amphipathic AChEs interact with phospholipid membranes. In a second major specifc aim, oligoDNA sequences corresponding to AChE mRNA segments for part of the RBC AChE polypeptides will be deduced from selected peptide sequences. These oligoDNAs will be chemically synthesized and labeled for use as probes for screening cDNA libraries. Identification of cDNAs for AChE mRNAs will provide a powerful tool for examining biosynthetic relationships among AChEs and for detailing the amino acid sequence differences among these AChEs.
A third aim i s to isolate AChE from mammalian brain and characterize any differences in the hydrophobic domain from that of RBC AChE.
A fourth aim i s focused on the collagen-like tail subunits from AChE isolated from eel electric organ. Attempts to obtain specific antisera to these subunits will be made, and chemical analyses to detect endogenous glycosaminoglycan associated with these subunits will be conducted. Glycosaminoglycan if present would likely play a major role in the attachment of AChE to the neuromuscular junction.
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