Choline acetyltransferase and acetylcholine levels in Drosophila melanogaster: A study using two temperature-sensitive mutants

Abstract

Choline acetyltransferase (ChAT, acetyl-CoA-choline-O-acetyltransferase, EC 2.3.1.6) activity was measured in homogenates prepared from wild type (Canton S) and two temperature-sensitive presumed ChAT structural gene mutants (Cha(ts1) and Cha(ts2); originally described by Greenspan, R. (1980) J. Comp. Physiol. 137: 83-92) of Drosophila melanogaster. Wild type flies grown at 32°C for 12 or 24 hr showed increased ChAT activity, whereas Cha(ts1) and Cha(ts2) flies showed a progressive decrease in enzyme activity at 32°C (restrictive temperature) when compared to flies reared at 18°C (permissive temperature). Acetylcholine (ACh) and choline levels were determined in formic acid-acetone extracts of individual fly heads, and the ACh levels showed the same variation with time at 32°C as did the ChAT activity. In contrast, choline levels did not vary in any regular pattern. Acetylcholinesterase (EC 3.1.1.7) activity did not vary during heat treatment (except for Chat(ts2) flies held at 32°C for 24 hr, where a decrease was observed) indicating that this treatment may be specific for ChAT. We conclude that ChAT activity is strongly correlated with ACh levels in Drosophila heads and may thus have an important regulatory role in determining the levels of ACh available for physiological function. We also report on the preliminary characterization of ChAT in both Cha(ts) mutants and compare the biochemical properties to those of wild type enzyme. Isoelectric focusing profiles of ChAT from both Cha(ts) mutants revealed enzymes with altered patterns compared to wild type, indicating that the mutations are most probably in the structural gene. The in vitro thermolability of individual isoelectric point forms of ChAT were similar to that of total ChAT activity in a homogenate. The k(m)'s for choline and acetyl-CoA were similar for enzyme from wild type and Cha(ts1) and differed only 2-fold for Cha(ts2). Immunoblot analysis of sodium dodecyl sulfate gels using a monoclonal antibody to Drosophila ChAT revealed only one 67,000-dalton band for either Cha(ts) mutant, whereas wild type enzyme showed two bands reacting with antibody, one at 67,000 and the other at 54,000 daltons. Since the monoclonal antibody directly inhibits enzyme activity, its is likely that the epitope it recognizes is at or near the active site of the enzyme.