İnsan Serum Bütirilkolinesterazının Olası Lipolitik Aktivitesi-Lipid Metabolizması ile İlişkisi.

Abstract

Cholinesterases (AChE and BChE) terminate acetylcholine-induced neurotransmission at cholinergic synapses in muscle-nerve junctions. We previously showed BChE expression is regulated by essential fatty acids. The similarity of catalytic domain of lipases with cholinesterases (ChE) led us to hypothesize that ChEs can hydrolyze various lipids and phospholipids. The possible hydrolysis of free fatty acids, triglycerides and membrane phospholipids such as phosphatidylcholine-sphingomyelin by cholinesterases was investigated, using chromatographic and various spectrophotometric-fluorometric methods. Lipolytic activity of BChE purified from human plasma, commercial BChE-AChE were compared with control enzymes from various sources (pancreatic (PanL) and wheat germ (BTL) lipase, phospholipase (PLA2), sphingomyelinase). PLA2 and sphingomyelinase activity of BChE were measured. PLA2 activity of BChE was partial, but there was no sphingomyelinase activity. Free fatty acid hydrolysis was studied with 4-mu palmitate as substrate. Results showed purified BChE can hydrolyze 4-mu palmitate as effectively as BTL. Michaelis-Menten kinetic results sort the affinity of the enzymes, that uses 4-mu palmitate as substrate, as follows: BTL (10.4 µM)> Pure BChE (34.2 µM)> PanL (129.8 µM)> tic BChE (186 µM). Trioleic acid (TO) hydrolysis of cholinesterases were analyzed by GC-MS and LC-MS/MS. According to GC-MS TO hydrolysis results, oleic acid released by BChE corresponded to 7% released by pancreatic lipase. LC-MS/MS results showed that TO hydrolytic activity of PanL (18593 ppm/min/mg protein enzyme) was higher than other enzymes (p<0.001). TO hydrolytic activity of purified BChE (714.3 ppm/min/mg protein enzyme), whose TO hydrolysis ability hasn’t been demonstrated to date, was higher than BTL (80.7 ppm/min/mg protein enzyme) (p=0.024). All studies were performed also with commercial AChE. However, AChE didn’t hydrolyze the substrates used here. The lipolytic activity of BChE in the presence of IsoOMPA, a specific inhibitor of BChE, and the supernatant obtained after BChE-treated lectin affinity chromatography was very low. This result shows the lipolytic activity belongs to BChE, and BChE hydrolyzes both trioleic acid and 4-mu palmitate.