Acetylcholinesterase isozymes from the housefly brain

Abstract

1. 1. Cholinesterase activity in insect head extracts was investigated by disc electrophoresis followed by spectrophotometric analysis. The supernatant of 100 000 × g, 1 h, revealed the presence of four isozymes in the housefly, two in the American cockroach and only one in the southern army worm. 2. 2. The four isozymes, present in the head extract of houseflies, were totally inhibited by 10 μM guthoxon or eserine but were unaffected by 10 μ M p- chloromercuribenzoate (PCMB) or tri-o- cresylphosphate . All four isozymes had higher activity with acetylthiocholine than butyrylthiocholine as substrates; indicating they are isozymes of acetylcholinesterase. 3. 3. Centrifugation at 200 000 × g for 4 h precipitated 66% of the acetylcholin-esterase originally present in the supernatant of 100 000 × g for 1 h but the four isozymes remained soluble and were estimated to have molecular weights of less than 500 000. 4. 4. Two methods of gel staining were used to detect esterase activity, the direct-coloring thiocholine method using acetylthiocholine as a substrated and the modified Gomori method using α-naphthylacetate. The first proved to be effective for detecting cholinesterase activity; but at least two of the eserine-sensitive bands found by the technique of Gomori 20 were not cholinesterase.