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Purification and some properties of house cricket (Acheta domesticus) acetylcholinesterase

1974; Elsevier BV; Volume: 4; Issue: 3 Linguagem: Inglês

10.1016/0020-1790(74)90049-3

1879-2928

An-horng Lee, Robert L. Metcalf, C. W. Kearns,

Insect Utilization and Effects

Abstract Bacterial protease from Bacillus subtilis was found to be effective in bringing the particulate house cricket head acetylcholinesterase (AChE) into solution. Following the solubilization, the AChE solution was further purified by DEAE-cellulose, Sephadex G-200, and hydroxylapatite column chromatography. A one-hundred-and-fifty fold purification with a specific activity of 9 μmoles per mg. protein per min. of the enzyme was obtained. There are at least two esterhydrolyzing enzymes in the crude homogenates of house cricket heads. One is the AChE which hydrolyses acetylcholine, phenyl acetate, and their thio-analogue, and is sensitive to low concetrations of physostigmine. Another enzyme utilizes phenyl acetate and phenylthioacetete as substrates and is insensitive to physostigmine. House cricket AChE hydrolyses acetylcholine and its thio-analogue at higher rates than other choline esters. In addition to the acetyl group the enzyme can only accommodate up to the propionyl group on the acid moiety of the choline esters. A series of K m values was determined for this enzyme as follows: acetylcholine 4.32 × 10 −5 M, acetylthiocholine 1.67 × 10 −4 M, propionylcholine 1.54 × 10 −4 M, propionylthiocholine 5.00 × 10 −5 M, phenyl acetate 4.76 × 10 −4 M, phenylthioacetate 1.43 × 10 −3 M. The anionic site of the enzyme seems to be important for the binding of phenyl acetate and phenyl-thioacetate. Maximal velocities for the hydrolysis of choline esters by this enzyme were shown at pS 3.4, pH 8.0. The sedimentation coefficient was determined by the sucrose density gradient centrifugation method as 4.66 S. The molecular weight was estimated as 6.62 × 10 4 .