RESUMEN
Choline acetyltransferase was demonstrated in nettles (Urtica dioica), peas (Pisum sativum), spinach (Spinacia oleracea), sunflower (Helianthus annuus) and blue--green algae by using a Sepharose--CoASH affinity column. The column effected a 1500-fold purification of the enzyme from nettle homogenates and was required for demonstrating activity in the other higher plants. Demonstration of the enzyme in blue-green algae suggests that acetylcholine was a biochemical necessity in the earliest photosynthetic organisms.
Asunto(s)
Acetilcolina/biosíntesis , Colina O-Acetiltransferasa/metabolismo , Plantas/enzimología , Animales , Cromatografía de Afinidad , Cianobacterias/enzimología , Fabaceae/enzimología , Cabeza/enzimología , Helianthus/enzimología , Moscas Domésticas/enzimología , Plantas MedicinalesRESUMEN
1. Polyacrylamide gel electrophoresis in Tris/glycine buffer (pH 8.3) revealed five forms of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) in the 100 000 X g, 1-h supernatants of aqueous fly-head extracts from the DDT/S strain. Five other housefly strains (CSMA, Bayer 21/199, Cradson/P, Malathion/R and DDT/R)were shown qualitatively to have the same soluble forms of the enzyme. 2. Plots of the electrophoretic mobility versus polyacrylamide concentration indicated that the multiple forms constituted a size isomer family. From the retardation coefficients derived from these plots, molecular weight estimates were obtained; these suggested that the smallest active component was a form of approx. 80 000 daltons. The higher aggregates, however, did not appear as simple oligomers of this component. 3. Density gradient sedimentation supported the electrophoretic findings. The smallest active component, with a sedimentation coefficient of 5.3 S, was confirmed as a molecular species of acetylcholinesterase that has not previously been obtained from house-flies; higher aggregates gave sedimentation coefficients of 7.4, 7.8. 8.1, and 11.8 S. 4. Gel-filtration on calibrated Sephadex G-150 columns provided further evidence that the smallest active component was a form of about 80 000 daltons. 5. Autolysis converted much of the particulate enzyme and all of the soluble forms into a species of approx. 160 000 daltons indistinguishable from the native 7.4-S form. Both the autolysed enzyme and the native 7.4-S form were susceptible to cleavage by disulphide reducing agents, and released catalytically active subunits that corresponded to the 5.3-S form of 80 000 daltons. The data were compatible with a monomer-dimer relationship between the 5.3-S and 7.4-S forms. 6. The possibility is suggested that a form of molecular weight approx. 80 000 constitutes the "fundamental unit" of insect cholinesterase.
Asunto(s)
Acetilcolinesterasa/análisis , Moscas Domésticas/enzimología , Cromatografía en Gel , Disulfuros , Electroforesis en Gel de Poliacrilamida , Cabeza/enzimología , Mercaptoetanol , Peso Molecular , Conformación ProteicaRESUMEN
1. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) of house-fly head tissue was solubilized as a 7.4-S form by autolysis for 80-100 h at 25 degrees C and pH 8.0. 2. The autolysed enzyme was purified by affinity chromatography, firstly on Con-A-Sepharose and subsequently on m-trimethylammoniumaniline-Affi-Gel 202. This sequence permitted overall purification yields of approx. 50% of the solubilized enzyme. 3. The 7.4-S purified enzyme was essentially homogeneous on polyacrylamide gel electrophoresis, and its specific activity coincided with the highest previously reported for fly-head acetylcholinesterase. 4. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and beta-mercaptoethanol revealed two major polypeptide components of molecular weight 82 000 and 59 000. Each of these polypeptides contained diisopropylphosphofluoridate-binding sites, as shown with [3H] diisopropylphosphofluoridate. 5. The results suggest a strong structural similarity between fly-head acetylcholinesterase and the purified electric eel enzyme.