Effect of specific inhibitors on cholinesterase activity in water extracts of selected basidiomycetes

Enzim kolinesteraza se nalazi u kolinergičnim sinapsama životinja i razgrađuje neurotransmiter acetilkolin te tako zaustavlja prijenos živčanog signala među stanicama. U zadnjem su desetljeću otkrivene i mnoge druge uloge tog enzima, na primjer uloga u staničnoj diferencijaciji, u komunikaciji među stanicama te u imunskom odgovoru. Enzim je bio nađen i u nekim gljivama kao npr. Aspegillus niger i Physarum polycephalum, no objave s tog područja vrlo su rijetke i ne postoje nikakva komparativna istraživanja o pojavnosti tih enzima u različitim vrstama nižih (askomicete) i viših (bazidiomicete) gljiva. U okviru diplomskog rada određivali smo aktivnost enzima kolinesteraza pomoću klasičnog kolorimetrijskog testa (Ellmanova metoda). Pomoću prethodno odabranog optimalnog supstrata napravili smo seriju mjerenja u ekstraktima bazidiomiceta koji su prethodno pokazali kolinesteraznu aktivnost. Prisutnost kolinesteraza u nekim ekstraktima potvrdili smo upotrebom specifičnih inhibitora. Nadalje smo provjerili da li i sami ekstrakti gljiva sadrže inhibitore koji mogu inhibirati životinjsku acetilkolinestarezu. Ekstrakte smo testirali i na prisutnost srodnih hidrolitičkih enzima lipaza. Potvrdili smo kako se u našim aktivnim uzorcima nalaze acetilkolinesteraze na koje djeluuju određeni inhibitori (diazinon, malation, triklorfon, etopropazin hidroklorid, neostigmin bromid, edrofonijev klorid, BW284C51), dok drugi inhibitori (Iso-OMPA, propidijev jodid) nisu imali djelovanje na aktivnost ovog enzima. Pretpostavljamo da u nekim uzorcima ima prisutnosti i butirilkolinesteraza, ili nekih drugačijih oblika acetilkolinesteraza. Aktivnost koja se pokazala u ovim uzorcima govori o tome da acetilkolinesteraza ima još funkcija osim one u živčanom prijenosu.The enzyme acetylcholinesterase is found in cholinergic synapses of animal species where it breaks down the neurotransmiter acetylcholine. In this way, it terminates the transmission of nerve signals. In the last decade some other roles of this enzyme have been discovered, for instance a role in cell differentiation, in communication between cells and in immune response. This enzyme has been also found in some lower fungi such as Aspegillus niger and Physarum polycephalum, but publications are rare in this field and there are no comparative studies between other species of lover (ascomycetes) and higher (basidiomycetes) fungi. In this master thesis, we determined the cholinesterase activity using a classic colorimetric test (Ellman method). We made a series of measurements in extracts of basidiomycetes which had shown cholinesterase activity in previous tests. We confirmed the presence of cholinesterases in some of these extracts using specific inhibitors. We also tested whether the extracts of mushrooms themselves have inhibitors that would inhibit cholinesterases of animal origin. We also tested the extracts for the presence of lipases. We confirmed the presence of acetylcholinesterases in the selected samples. Some inhibitors, such as (Diazinon, malathion, trichlorfon, neostigmine bromide, edrophonium chloride, ethopropazine hydrochloride, BW284C51) inhibited the extracts, while some (Iso-OMPA, propidium iodide) had no effect. We suggest that in some extracts also cholinesterases with butyrylcholine binding activity are present. The activity shown in the analysed samples suggests that there are more functions to acetylcholinesterase except for the ones in transmission of nerve signal