Purification, Some Characterization And Modification Of Groups Of Acetylcholinesterase From Porcine Brain

The neurotransmitter acetylcholine and cholinergic system containing acetylcholine synthesis enzymes, two kinds of acetylcholine receptors and acetylcholinesterase, which widely existing in plants and animals, involves in regulation of some important physiological processes. AChE is a kind of hydrolase, and plays an important role in animal nerve conduction. It can effectively the neurotransmitter acetylcholine hydrolyzed into acetic acid and choline, so as to ensure the normal transmission of nerve impulses in the synapse. It also has a non-catalytic functions which involves in the development of cells maturation, migration, adhesion and apoptosis, promoting neuronal growth and nerve regeneration, blood cell formation, inflammation and immune responses. AChE can be generally applied in environmental protection, medicine, agriculture, food, military and other fields. The source of AChE in China has been dependent on imports, the imported enzyme is expensive, and the course of importing needs much time. To find a cheap source of enzyme is very important, therefore, it is necessary to strengthen the study on purification of AChE. We have isolated and purified AChE from porcine brain and researched on the partial properties of the enzyme. Our works aimed on finding a pathway to obtain AChE and provide the data for deeply investigation of AChE and application of porcine brain. The results are as follows:1. Purification of acetylcholinesterase from porcine brainFresh porcine brain was selected as a source of enzyme, and the extraction and purification of AChE by means of centrifugal, sulfate precipitation, ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose and gel filtration chromatography on Superdex-200.The enzyme was purified to electrophoretic homogeneity. It was purified26.97-fold and the activity recovery11.95%was obtained. Its specific activity was2.05U/mg. 2. The Characterization of acetylcholinesteraseThe relative molecular weight of this acetylcholinesterase was257.30kD, and the weight of subunit was66.94kD.The acetylcholinesterase consists four idenitcal subunits. The optimum pH and temperature of the enzyme for the hyrolysis of S-Acetylthiocholine were7.4and37℃. The enzyme was stable in the pH ranges of6-8and at temperatures below40℃. It could be inhibited by excess substrate when the concentration is higher than4.0mmol/L. The apparent Km of the enzyme was0.96mmol/L at pH7.4and3℃.3. The effect of organic solvents, compounds and metal ions on the enzymeEthanol, isopropanol, SDS, hydrogen peroxide, ascorbic acid, oxalic acid and urea can significantly inhibit the activity of this enzyme. And low concentration of EDTA-Na2and Mg2+had activation effect on it. Ba2+and Zn2+had strong inhibition on this enzyme.4. Research on the functional groups of the enzymeWe analyzed the enzyme’s functional groups modified with chemical modification methods. The results showed that cysteine thiol group, methionine residue, histidine imidazole, and serine residue may be the essential groups of the enzyme active site and participate in the composition of the active center. Disulfide bond may maintain activity of acetylcholinesterase from porcine brain. However, arginine guanidine, phenolic hydroxyl group of tyrosine, Lysine residue and tryptophan residues may not be the necessary for the activity of acetylcholinesterase and they may not participate in the active center of acetylcholinesterase.