Napredna pretraga

Pregled bibliografske jedinice broj: 147937

Kinetics of ethopropazine binding to butyrylcholinesterase in the absence and presence of acetylthiocholine


Reiner, Elsa; Šinko, Goran; Bosak, Anita; Simeon-Rudolf, Vera; Radić, Zoran; Taylor, Palmer; Stojan, Jure; Goličnik, Marko
Kinetics of ethopropazine binding to butyrylcholinesterase in the absence and presence of acetylthiocholine // Cholinesterases in the Second Millennium : Biomolecular and Pathological Aspects / Inestrosa, Nibaldo C. ; Campos, Eliseo O. (ur.).
Santiago de Chile: Universidad Catolica de Chile, 2004. str. 187-189


Naslov
Kinetics of ethopropazine binding to butyrylcholinesterase in the absence and presence of acetylthiocholine

Autori
Reiner, Elsa ; Šinko, Goran ; Bosak, Anita ; Simeon-Rudolf, Vera ; Radić, Zoran ; Taylor, Palmer ; Stojan, Jure ; Goličnik, Marko

Vrsta, podvrsta i kategorija rada
Poglavlja u knjigama, znanstveni

Knjiga
Cholinesterases in the Second Millennium : Biomolecular and Pathological Aspects

Urednik/ci
Inestrosa, Nibaldo C. ; Campos, Eliseo O.

Izdavač
Universidad Catolica de Chile

Grad
Santiago de Chile

Godina
2004

Raspon stranica
187-189

ISBN
956-299-127-X

Ključne riječi
Inhibition, ethopropazine, butyrylcholinesterase

Sažetak
Ethopropazine is a potent reversible inhibitor of butyrylcholinesterase (BChE). The nature of its binding site(s) on the enzyme is not completely understood. We compare in this study the enzyme-inhibitor dissociation constants Ki derived from the kinetics of competition between ethopropazine and substrate (acetylthiocholine ; ATCh) with the enzyme-inhibitor dissociation constants Kd derived in the absence of substrate. Two schemes A (cf.1) and B (cf.2) for the enzyme-inhibitor-substrate interactions were applied to evaluate Ki. The Kd values were derived from the ratio of the rate constant of dissociation (koff) and rate constant of association (kon) of ethopropazine from and to BChE. The BChE activities were measured spectrophotometrically (by conventional and stopped-flow techniques) with the thiol reagent DTNB. Intrinsic fluorescence quenching of BChE tryptophans was measured to evaluate kon and koff. Partially purified horse serum BChE and racemic ethopropazine (20-20000 nM) were used. The range of ATCh concentrations was 0.005-50 mM (scheme A) and 0.02-50 mM (scheme B). Applying scheme A, two Ki constants were obtained at 37 oC, 230 (for IE) and 1320 (for ISE) nM, and three constants at 25 oC, 160 (for IE), 450 (for ISE) and 4400 (IIE) nM (3). According to that scheme the binding sites for ethopropazine (I) and ATCh (S) can be either the non-productive peripheral site or the catalytic site. Applying scheme B, only one Ki constant was obtained at each temperature: 720 nM at 37 oC (4) and 200 nM at 25 oC. These constants were attributed to binding of ethopropazine to the non-productive peripheral site of BChE, because competition between ethopropazine and ATCh was pronounced at substrate concentrations approaching the Kss value for ATCh. Determination of Kd values similarly did not reveal more than one binding site at each studied temperature: 91 nM at 37 oC and 77 nM at 23 oC. From the outlined comparisons it seems that the number and location of binding sites for ethopropazine are still not clarified. However, two different approaches (evaluation of Kd and evaluation of Ki from scheme B) both indicate only one binding site on BChE. The affinity of ethopropazine for that site appears to be lower when evaluated in the absence of substrate. Increasing the temperature from 23 or 25 oC to 37 oC affected minimally the Kd values and the Ki for IE (scheme A), while the other Ki constants increased three-fold or more. References: (1) Stojan et al., FEBS Lett. 440 (1998) 85-88 ; (2) Simeon-Rudolf et al., Croat. Chem. Acta 74 (2001) 173-182 ; (3) Goličnik et al., Arch. Biochem. Biophys. 398 (2002) 23-31 ; (4) Reiner et al., Proceedings of the "Cholinergic Mechanisms Symposium", St. Moritz, Switzerland (2002), in press.

Izvorni jezik
Engleski

Znanstvena područja
Kemija



POVEZANOST RADA


Projekt / tema
0022014

Ustanove
Institut za medicinska istraživanja i medicinu rada, Zagreb