Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis)
| Autor: | Elvio Giovannini, Vincenzo Nicola Talesa, Marta Grauso, Gabriella Rosi, Jean-Pierre Toutant |
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| Přispěvatelé: | Différenciation Cellulaire et Croissance (DCC), Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2) |
| Rok vydání: | 1995 |
| Předmět: |
[SDV]Life Sciences [q-bio]
Biochemistry Substrate Specificity Affinity chromatography Leeches Animals [INFO]Computer Science [cs] Molecular Biology ComputingMilieux_MISCELLANEOUS chemistry.chemical_classification Chromatography BIOCHIMIE Molecular mass biology Substrate (chemistry) Cell Biology biology.organism_classification Enzyme Activation Hirudo medicinalis Enzyme chemistry Sephadex Concanavalin A Acetylcholinesterase biology.protein Propionate Research Article |
| Zdroj: | Biochemical Journal Biochemical Journal, Portland Press, 1995, 306, pp.687-692 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj3060687 |
| Popis: | Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo medicinalis). A ‘spontaneously-soluble’ portion of AChE activity (SS-AChE) was recovered from haemolymph and from tissues dilacerated in low-salt buffer. A second portion of AChE activity was obtained after extraction of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogeneity by affinity chromatography on edrophonium- and concanavalin A-Sepharose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native SS-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedimenting at 5.0 S in sucrose gradients with or without Triton X-100, suggesting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-specific phospholipase C suppressed aggregation and gave a 7 S peak. DS-AChE was thus an amphiphilic glycolipid-anchored dimer. Substrate specificities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-AChE displayed only limited variations in Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. By contrast, DS-AChE displayed higher Km values with uncharged than with charged substrates. SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M. |
| Databáze: | OpenAIRE |
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