Photodestruction of acetylcholinesterase.

Abstract

Ultraviolet irradiation of 11I acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) produces a loss of trytophan fluorescence which is best descri as the sum of two separable first-order processes, one much more rapid than the other. In addition, the enzyme undergoes an all-or-none inactivation that is monotonically first order. Simultaneous with activity loss, photoscission takes place and results in a molecular weight drop of 1 X 105; this decrease is first order with a rate constant identical to that for enzymatic inactivation. These processes are accompanied by apparent conformational changes, as shown by circular dichroic and difference absorption spectra. The relative photochemical inactivation efficiency of incident light is unity when corrected for the wavelength dependence of fluorescence excitation, which is consistent with an efficient F6rster resonance. transfer of energy among the aromatic chromophores. The extreme sensitivity of acetylcholinesterase to photodestruction upon photon absorption and the several events that follow it not only suggest that these findings might be a basis for a useful molecular probe of the structure of'this enzyme, but also indicate that additional care should be taken when conducting spectroscopic studies in the UV region.Recent studies on acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) have used extrinsic fluorescent probes to determine active-site geometry (1-3). However, little work has been done on the protein's intrinsic fluorescence. During such a study on llS acetylcholinesterase, we observed that the enzyme was denatured by incident UV irradiation. Accordingly, we performed some preliminary characterizations of this process, and we report here our initial findings.MATERIALS AND METHODS Homogenous 11S acetylcholinesterase was prepared from Electrophorus electricus and its activity was assayed as described (4). Irradiation and fluorescence measurements were done at 250C in 1 X 1 cm-quartz cells with a thermostatted Perkin-Elmer MPF-44B fluorometer operated in the quantum corrected mode. Circular dichroic spectra were obtained at room temperature (--200C) with a JASCO (Tokyo, Japan) J-500 A recording spectropolarimeter standardized with D-10-camphorsulfonic acid (5). Difference absorption measurements were made at 250C with a Beckman model 25 spectrophotometer. Molecular weight determinations were carried out as before at SOC and 14,000 rpm in 4-mm solution columns (6) with an initial A2w of 0.2. A iv of 0.731 cm3/g was used for control and irradiated enzyme.Theoretical calculations of concentration, C, as a function of radial distance for model systems were made as follows:[2] in which A = (1 -ip) w2/2RT (the symbols having their customary meanings), a and b are meniscus and solution base radius, respectively, Mt is the molecular weight of the ith species, Co,j, the starting concentration of the ith species, and Ca., and Cj are the concentrations of the ith species at the meniscus and radius positions a and r, respectively. Th...

Fish-mediated trait compensation in zooplankton

“…The negative effect of UVR on macromolecules such as ChE may be either caused by direct effects of radiation on the molecule (Bishop et al. 1980) or an indirect effect through UVR‐generated ROS that in turn oxidize labile molecules (Lesser 2006; Souza et al.…”

“…The negative effect of UVR on macromolecules such as ChE may be either caused by direct effects of radiation on the molecule (Bishop et al 1980) or an indirect effect through UVR-generated ROS that in turn oxidize labile molecules (Lesser 2006;Souza et al 2010a). Carotenoids protect organisms from UVR in both ways, as they act as sunscreen absorber (Garcı´a-Pichel 1994), as well as ROS quencher (Lesser 2006).…”

Effect of Ultraviolet Radiation on Acetylcholinesterase Activity in Freshwater Copepods

“…In particular, AChE inhibition is an indicator of exposure to organophosphorus and carbamate pesticides, toxins, and toxic metals such as cadmium, lead and copper (15) and is considered as an early warning signal of ecotoxicological risk in marine zooplankton (16). However, natural factors such as UVR probably influence this enzyme activity, but so far the only available data come from an in vitro study which indicates that AChE was denatured by incident UVR with loss of enzyme activity (17). Based on this early evidence we can also postulate a direct effect of UVR on the enzyme activity in planktonic organisms.…”

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