Purification of Acetylcholinesterase from Sea Urchin (Hemicentrotus pulcherrimus) Embryos by Affinity Chromatography

Abstract

Only one form of acetylcholinesterase (AchE) was detected in Hemicentrotus pulcherrimus embryos. In H. pulcherrimus embryos as well as in the other sea urchin embryos, AchE activity begins to increase rapidly after gastrula stage.Purification of AchE from plutei has been carried out by the procedure including affinity chromatography. Purified AchE had the activity 14,600 times higher than that of homogenate, and the final yield of AchE was 8%. The enzyme seems to be electrophoretically homogeneous, and has a molecular weight of 3 x lo5 as determined by Sepharose CL6B column chromatography.In sea urchin embryos, AchE activity is little detectable before gastrula stage, and at this stage AchE activity begins to increase sharply (1, 2). However, it is not known whether the sudden appearance of this enzyme is due to the onset of transcription of AchE gene or due to the onset of translation of the maternal stored mRNA of AchE. WHITTAKER (3) and SATOH and IKEGAMI (4, 5) presented the evidences suggesting that acidian embryos express the AchE gene activity at the certain time of development and that the embryonic cells seems to determine the timing of the expression of this gene seems by somehow measuring the number of DNA replication cycles.As the first step for the elucidation of the molecular mechanism underlying the production of this enzyme by gastrula cells, we attempted to purify AchE from sea urchin embryos. Previously the partial characterization of sea urchin AchE has been reported by others using the partially purified enzyme preparation (2). In the present paper, we describe a procedure for the preparation of AchE from sea urchin embryos. The procedure contains the affinity chromatography on a trimethyl (p-aminophenyl) ammonium-Sepharose (TMAPA-Sepharose) column developed by BERMAN and YOUNG (6) for the purification of AchE from eel electric tissue and bovine erythrocytes. MATEFUALS AND METHODS Preparation of embryoEggs and sperm of Hemicentrotus pulcherrimus were collected by artificial spawning induced by a KCl solution. Inseminated eggs were raised in filtered sea water at 20°C with a constant stirring. Embryos were harvested by centrifugation, and kept frozen at -20°C until use. Enzyme assayThe activity of AchE was assayed by the method of Ellman et al. (7), using 2.3 mM acetylthiocholine iodide as a substrate.For the determination of ontogenic change of the enzyme activity, los embryos were suspended in 5 ml of ice-cold deionized water and sonicated by a Branson Sonifier at 40 W for 30 sec. An aliquot of the sonicate was 85

Acetylcholinesterase in the sea urchin Lytechinus variegatus: Characterization and developmental expression in larvae

“…AChE activity has been traced throughout the development of several species of sea urchins. These species include P. (Ozaki, 1974), S. purpuratus (Ozaki, 1976), and Hemicentrotus pulcherrimus (Akasaka et al, 1986). Not only is AChE activity present in sea urchin embryos and larvae, a characteristic trend of increasing activity throughout development is observed in various species reported in the literature.…”

“…Not only is AChE activity present in sea urchin embryos and larvae, a characteristic trend of increasing activity throughout development is observed in various species reported in the literature. ACh and AChE are present in early cleavages of the developing embryo, but sustained increases in the levels of ACh and AChE activity are observed during gastrulation (Falugi et al, 2002;Akasaka et al, 1986) and post-gastrulation (Augustinsson and Gustafson, 1949;Ozaki, 1974;1976). This rapid increase in AChE activity is thought to be the possible beginnings of neuronal differentiation (Akasaka et al, 1986).…”

“…ACh and AChE are present in early cleavages of the developing embryo, but sustained increases in the levels of ACh and AChE activity are observed during gastrulation (Falugi et al, 2002;Akasaka et al, 1986) and post-gastrulation (Augustinsson and Gustafson, 1949;Ozaki, 1974;1976). This rapid increase in AChE activity is thought to be the possible beginnings of neuronal differentiation (Akasaka et al, 1986). Through the use of an AChE staining method, the Cu-thiocholine method of Karnovsky and Roots (1964), Ozaki (1974;1976) determined that AChE is localized in the mesenchyme cells of sea urchin larvae.…”

Developmental abnormalities and neurotoxicological effects of CuO NPs on the black sea urchin Arbacia lixula by embryotoxicity assay

“…AChE plays a key role during the migration of PMCs and spicule elongation (Ohta et al, 2009). Indeed, AChE activity is necessary for the development of several M. Maisano et al / Marine Environmental Research xxx (2015) 1e7 5 species of sea urchins including Paracentrotus depressus (Ozaki, 1974), Strongylocentrotus purpuratus (Ozaki, 1976), Hemicentrotus pulcherrimus (Akasaka et al, 1986). In embryos, AChE level increases during gastrulation and post-gastrulation, as it is probably involved in the neuronal differentiation (Akasaka et al, 1986;Ohta et al, 2009).…”

“…Indeed, AChE activity is necessary for the development of several M. Maisano et al / Marine Environmental Research xxx (2015) 1e7 5 species of sea urchins including Paracentrotus depressus (Ozaki, 1974), Strongylocentrotus purpuratus (Ozaki, 1976), Hemicentrotus pulcherrimus (Akasaka et al, 1986). In embryos, AChE level increases during gastrulation and post-gastrulation, as it is probably involved in the neuronal differentiation (Akasaka et al, 1986;Ohta et al, 2009). AChE is known to be an essential enzyme for cholinergic neurotransmission in both vertebrates and invertebrates, including sea urchin, and its measurement is used as biomarkers of neurotoxicity in several environmental studies (Cappello et al, 2015;D'Agata et al, 2014b;De Domenico et al, 2013;Fasulo et al, 2010a;Matozzo et al, 2005).…”