Localization and Multiple Forms of Acetylcholinesterase in Sea Urchin Embryos

Ozaki, Hironobu

doi:10.1111/j.1440-169x.1974.00267.x

Cited by 39 publications

(32 citation statements)

References 24 publications

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“…1) as pointed out previously (1,2). Only a weak AchE activity was found in the embryo before blastula stage.…”

Section: Ontogenic Change Of Ache Activitymentioning

confidence: 48%

“…In sea urchin embryos (P. depressus), the presence of two forms of AchE have previously been reported through the electrophoretic analysis of crude enzyme preparation (2). However, only one major active band of esterase was detected in the electrophoretogram of the crude enzyme preparation, when the gels were stained under the conditions described in the text.…”

Section: Discussionmentioning

confidence: 95%

“…By the histochemical studies, it has been demonstrated that the enzyme is mainly localized in the mesenchyme cells associated with the spicules, the edge of the oral lobe and the anal arms (2), which seem to be subjected to innervation. Since the enzyme activity is low before blastula stage, and then begins to increase thereafter (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

et al. 1986

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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

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“…1) as pointed out previously (1,2). Only a weak AchE activity was found in the embryo before blastula stage.…”

Section: Ontogenic Change Of Ache Activitymentioning

confidence: 48%

Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

et al. 1986

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“…This enzyme has been detected in homogenates of embryos of several species of echinoderms (2, 25,26,29) and amphibians (3 1,37). Acetylcholinesterase activity has been identified cytochemically in primary and secondary mesenchymal cells in sea urchin embryos (22).…”

mentioning

confidence: 99%

ACETYLCHOLINE AND ACETYLCHOLINESTERASE ACTIVITY IN EARLY EMBRYOS OF THE MEDAKA ORYZIAS LATIPES, A TELEOST

Fluck¹

1978

Dev Growth Differ

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Acetylcholinesterase (AChE) activity is present in homogenates of medaka embryos during cleavage and epiboly. The levels of AChE activity change little during this period of development and are similar in embryos grown at either 15°C or 25°C. The specific activity of AChE in cells isolated from blastulae is 0.06 mmoles substrate hydrolyzed/min/g protein, a value comparable to that of chicken myoblasts and myotubes in vitro. Acetylcholinesterase activity was detected cytochemically in all cells dissociated from blastulae and gastrulae. In deep blastomeres AChE activity is present nearly throughout the cytoplasm; it is absent from peripheral regions of the cytoplasm which are involved in circus, or limnicolor, movements. Acetylcholine-like activity in extracts of the embryos was assayed using the clam heart ventricle bioassay. The active principle, which caused a decrease in both the frequency and magnitude of ventricular contractions, was inactivated by heating at pH 10 and by incubation at pH 7.0 with commercial AChE. The effect of the active principle on the clam heart was blocked by mytolon chloride, a drug which specifically blocks the effect of acetylcholine on the clam heart. The active principle migrated on Whatman #1 chromatography paper with the same R, as authentic acetylcholine in three solvent systems. The amount of acetylcholine in blastulae is about 4 picomoles/embryo.

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Two cholinesterase activities and genes are present in amphioxus

et al. 1997

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To obtain information about the evolution of the cholinesterases, acetylcholines‐terase (AChE) and butyrylcholinesterase (BuChE) in the vertebrates, we investigated the cholinesterase (ChE) activity of the cephalochordate amphioxus (Branchiostoma floridae and Branchiostoma lanceolatum). On the basis of evidence from enzymology, pharmacology, and molecular biology, we conclude that amphioxus possesses two ChE activities and two ChE genes. Two covalent inhibitors of cholinesterases were able to pharmacologically isolate the two activities as drug‐sensitive ChE and drug‐resistant ChE. Kinetically, in terms of substrate specificity, the drug‐sensitive ChE resembles vertebrate AChE, and the drug‐resistant ChE resembles the BuChE of cartilaginous and bony fish or the intermediate ChE of protostome invertebrates. We also used the polymerase chain reaction with degenerate oligonucleotide primers and genomic DNA to obtain clones of 1,574 and 1,011 bp corresponding to two cholinesterase genes from amphioxus, which we designated as ChE1 and ChE2. ChE2 codes for an enzyme with an acyl‐binding pocket sequence, a portion of the protein that plays an important role in determining substrate specificity, typical of invertebrate ChE. ChE1, which contains a 503‐bp intron, encodes a protein with a novel acyl binding site. Phylogenetic analysis of the sequences suggests that the two genes are a result of a duplication event in the lineage leading to amphioxus. We discuss the relevance of our results to the evolution of the cholinesterases in the chordates. Previously, we reported that amphioxus contained a single cholinesterase activity with properties intermediate to AChE and BuChE (Pezzementi et al. [1991] In: Cholinesterases: Structure, Function, Mechanism, Genetics and Cell Biology. J. Massoulié et al., eds. ACS: Washington, D.C., pp. 24–31). J. Exp. Zool. 277:213–229, 1997. © 1997 Wiley‐Liss, Inc.

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Localization and Multiple Forms of Acetylcholinesterase in Sea Urchin Embryos

Cited by 39 publications

References 24 publications

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

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

ACETYLCHOLINE AND ACETYLCHOLINESTERASE ACTIVITY IN EARLY EMBRYOS OF THE MEDAKA ORYZIAS LATIPES, A TELEOST

Two cholinesterase activities and genes are present in amphioxus

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