Preliminary characterization of cholinesterase activity in wheat

The purpose of this study was to compare and characterize the enzymatic activity directed towards two substrates, acetylthiocholine and indophenyl acetate. Cholinesterease activity is defined as the enzymatic hydrolysis of acetylthiocholine; IPAse activity is defined as the enzymatic hydrolysis of indophenyl acetate. The responses of both assays to changes in temperature, pH, and substrate concentration were determined. In addition, the effects of inhibitors, metal ions, and different substrates on the two activities were examined. The enzymatic activities were also partially purified.

It was found that as temperature increased above 30 C, enzymatic activity measured by both assays decreased. For the IPA assay, the pH optimum was 7.2; for the ATCH assay, the pH optimum was 8.2. The K_m’s for IPA, ATCH, PTCH, and BTCH were 1.67 x 10^-4 M, 4.4 x 10^-4 M, 2.0 x 10 M, and 1.60 x 10^-4 M, respectively. Choline and acetylcholine did not inhibit the IPA assay. Inhibition of the ATCH assay by AMO-l618, eserine, and neostigmine occurred at 10^-5 M concentrations. Eserine, AMO- 1618, and neostigmine inhibited the IPA assay only at relatively high concentrations; PCMPs did not inhibit the IPA assay. All of the metal ions used, Ca²⁺ , Mn²⁺ , Mg²⁺ , Li²⁺ , Co²⁺ , Zn²⁺ , Fe²⁺ , and Cu²⁺ were found to inhibit the ATCH assay, while Mn²⁺ , Mg²⁺ , Ly⁺, and C²⁺ were found to stimulate the IPAse activity. Using thiocholine esters as substrate, it was found that as the length of the side chain increased, the ability for enzymes to cleave the substrates decreased.

Because the responses of the two assays differ in the presence of inhibitors and activators, it is thought that the assays are monitoring a complex mixture of hydrolytic activities.