MIPAFOX DIFFERENTIAL INHIBITION ASSAY FOR HEART-MUSCLE CHOLINESTERASES - SUBSTRATE-SPECIFICITY AND INHIBITION OF 3 ISOENZYMES BY PHYSOSTIGMINE AND QUINIDINE

1. A differential inhibition assay was developed for the quantitative determination of cholinesterase isoenzymes acetylcholinesterase (AChE; EC 3.1.1.7), cholinesterase (BChE; EC 3.1.1.8), and atypical cholines terase in small samples of left ventricular porcine heart muscle. 2. T he assay is based on kinetic analysis of irreversible cholinesterase i nhibition by the organophosphorus compound N,N'-di-isopropylphosphorod iamidic fluoride (mipafox). With acetylthiocholine (ASCh) as substrate (1.25 mM), hydrolytic activities (A) of cholinesterase isoenzymes wer e determined after preincubation (60 min, 25 degrees C) of heart muscl e samples with either saline (total activity, A(T)), 7 mu M mipafox (A (M1)), or 0.8 mM mipafox (A(M2)): (BChE)=A(T) - A(M1), (AChE)=A(M1) - A(M2), (Atypical ChE)=A(M2). 3. The mipafox differential inhibition as say was used to determine the substrate hydrolysis patterns of myocard ial cholinesterases with ASCh, acetyl-beta-methylthiocholine (A beta M SCh), propionylthiocholine (PSCh), and butyrylthiocholine (BSCh). The substrate specificities of myocardial AChE and BChE resemble those of erythrocyte AChE and serum BChE, respectively. Michaelis constants K-M with ASCh were determined to be 0.15 mM for AChE and 1.4 mM for BChE. 4. Atypical cholinesterase, in respect to both substrate specificity and inhibition kinetics, differs from cholinesterase activities of ver tebrate tissue and, up to now, could be identified exclusively in hear t muscle. The enzyme's Michaelis constant with ASCh was determined to be 4.0 mM. 5. The reversible inhibitory effects of physostigmine (eser ine) and quinidine on heart muscle cholinesterases were investigated u sing the differential inhibition assay. With all three isoenzymes, the inhibition kinetics of both substances were strictly competitive. The physostigmine inhibition of AChE was most pronounced K-i=0.22 mu M). Quinidine most potently inhibited myocardial BChE (K-i=35 mu M). (C) 1 997 Elsevier Science Inc.