PURIFICATION AND CHARACTERIZATION OF ACETYLCHOLINE ESTERASE FROM LIVER OF TILAPIA (TILAPIA ZILLI)

Abstract

Acetylcholinesterase (AChE, EC 3.1.1.7) is an enzyme that catalyzes the hydrolysis of acetylcholine into choline and acetic acid at cholinergic synaptic sites. AChE has been successfully separated, purified and characterized from many vertebrates and invertebrates prior to their use as tools in environmental assessment, but little work has been done on the acetylcholine esterase of the liver. With this in mind, this study is aimed at purifying and characterizing acetylcholine esterase from the liver of Tilapia zilli. The specific objectives of the research include the: purification of acetylcholine esterase using ion-exchange and gel-filtration chromatography; determination of physicochemical characteristics; determination of kinetic parameters of purified AChE and inhibition of purified AChE activity with some known insecticides. Acetylcholine esterase activity and protein concentration were determined according to standard procedures. The enzyme was purified to homogeneity by fractional ammonium sulphate precipitation (40 – 60 %), ion-exchange chromatography on DEAE- Sephadex A50 column and gel filtration chromatography on Sephadex G-200 column. The molecular weight of the AChE was also determined. The effects of varying pH and temperature on enzyme activity were carried out. The stability of the enzyme activity at different pH and temperature were evaluated. In addition, kinetics and inhibitory studies of purified acetylcholine esterase were carried out. The crude enzyme activity and protein concentration were 6.37 mmol/min/ml and 90 mg/ml respectively. The ion-exchange chromatography on DEAE- Sephadex A50 column gave two peaks, which means the AChE possibly has two iso-enzymes, giving 26-fold purification with yield of 21.32% and 39-folds purification with yield of 15.60% respectively for both iso-enzymes A and B. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed the molecular weight of the purified enzyme A to be 42 kDa. The two purified peaks A and B of acetylcholine esterase were active and stable over a wide pH range of 4 – 8, with optimum activity being observed at pH 8 for both isoenzymes. The purified isoenzymes were relatively stable: respectively retained about 55% of initial activity when incubated at 30 °C for 10 min. The apparent Km and Vmax were 0.5 mM and 25.1 mmol/min/ml for iso-enzyme A while 0.31 mM and 15.7 mmol/min/ml for iso-enzyme B respectively. Both isoenzymes were inhibited by the following insecticides: chlorpyrifos, cypermethrin and 2, 2, dichlorovinyl dimethyl phosphate. The results from this research showed that, the purified enzyme contained two isoenzymes, the Km revealed that isoenzyme B has higher affinity for the substrate than isoenzyme A and inhibition of enzyme activity was dependent on concentration of these inhibitors.