The overall objective of this project is to elucidate the role of the enzyme choline acetyltransferase in the regulation of the synthesis of the neurotransmitter acetylcholine. One of the primary specific goals of this project is to obtain a cDNA corresponding to ChAT messenger RNA. New cDNA libraries have been prepared in lambda gt10 and lambda Zap using polyA mRNA from the cholinergic cell line CHP134. These libraries will be screened with several anti-ChAT antisera prepared in this laboratory as well as with oligonucleotide probes prepared on the basis of protein sequence generated in this laboratory. To date we have sequenced a total of 10 cyanogen bromide and tryptic peptides corresponding to approximately 20% of the primary sequence of human ChAT. Isolated cDNA clones will be used to study the regulation of ChAT induction in cultured cells as well as for the expression of the enzyme for mechanistic studies. It has been shown that ChAT can be phosphorylated by a Ca/calmodulin kinase and protein kinase C from rat brain. Studies will also focus on determining the effect of phosphorylation on the properties of the enzyme. These studies will involve an analysis of the kinetics of Ca/calmodulin kinase phosphorylated enzyme in the presence and absence of activator protein and protein kinase C phosphorylated enzyme. Two pI forms of the rat brain enzyme will be isolated to determine whether they represent native and phospho enzyme. Studies with cultured cells will be used to present evidence for ChAT phosphorylation in vivo and to assess the effect of phosphorylation on acetylcholine synthesis. The isolation and sequencing of peptides containing active site cysteine, arginine and histidine residues are planned. The identification of these residues in the enzyme will provide the foundation with which to identify the active site and will serve as a focal point for site- specific mutagenesis.
