Purification of human red cell acetylcholinesterase

Zittle, Charles A.; Dellamonica, E. S.; Custer, J.H.

doi:10.1016/0003-9861(54)90303-3

Cited by 43 publications

(11 citation statements)

References 4 publications

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“…Erythrocyte AchE has never been purified, although partial purification procedures have been presented (4)(5)(6). The work outlined here was initiated to facilitate complete purification of both proteins prior to comparative studies of their molecular structures and function.…”

Section: And 2)mentioning

confidence: 99%

Rapid and Complete Purification of Acetylcholinesterases of Electric Eel and Erythrocyte by Affinity Chromatography

Berman

Young

1971

Proc. Natl. Acad. Sci. U.S.A.

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Affinity chromatography has been used to purify acetylcholinesterase both from the electric tissue of Electrophorus electricus and from bovine erythrocyte membranes. For this purpose, several specific enzymic inhibitors of each protein were synthesized and joined covalently to an insoluble support resin. AchE is selectively retained by such inhibitor-resins when highly impure solutions are chromatographed upon them. After removal from the resin, both enzymes are electrophoretically homogeneous and they may be recovered in yields of 75% or more.In the study to be presented below, we have used affinity chromatography to purify acetylcholinesterase (AchE) from both the electric tissue of Electrophorus electricus and from bovine erythrocyte membranes; only a single purification step is required. The central feature of this technique depends upon coupling a specific inhibitor of the enzyme to an inert, insoluble support resin. Under appropriate solvent conditions, chromatography of the enzyme upon the inhibitor-resin compound selectively retains AchE and thus eliminates contaminating molecules that possess no affinity for the inhibitor ligand. Subsequently, AchE is removed from the column by elution with another specific inhibitor whose inhibition constant (Ki) is less than that of the inhibitor-resin compound.Purification by affinity chromatography therefore requires (a) synthesis of an inhibitor that can be covalently bound to a solid support; (b) attachment of the inhibitor molecule to the resin support by a chain of atoms long enough to enable the inhibitor to interact readily with the binding site(s) of the enzyme; and (c) establishing conditions under which the protein can be selectively removed from the column. (For a summary of recent studies on affinity chromatography, see refs. 1 and 2.)In earlier studies, Leuzinger and Baker (3) have used conventional procedures to purify eel AchE. Erythrocyte AchE has never been purified, although partial purification procedures have been presented (4-6). The work outlined here was initiated to facilitate complete purification of both proteins prior to comparative studies of their molecular structures and function. MATERIALS AND METHODS Measurements of enzyme activityAchE activity was measured from the amount of 0.01 NNaOH needed to maintain constant pH during hydrolysis of acetylcholine at pH 7.5 and 22°C. The incubation solvent contained Abbreviation: AchE, acetylcholinesterase. For these studies, the nonionic resin agarose (Bio-Rad) was used as the insoluble support. 2% agarose (molecular weight exclusion limit = 5 X 107 g/mol) was selected so that AchE (MW = 250,000) (3) would potentially be able to interact with inhibitor molecules bound on both the inside and the outside of the gel matrix.Cuatrecasas (2) has recently presented chemical methods for attachment of small organic molecules to agarose. The several inhibitor ligands used in this study are illustrated in Table 1 and their syntheses are described below.Trimethyl (p-aminophenyl)ammonium chloride hydro...

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Section: And 2)mentioning

confidence: 99%

Rapid and Complete Purification of Acetylcholinesterases of Electric Eel and Erythrocyte by Affinity Chromatography

Berman

Young

1971

Proc. Natl. Acad. Sci. U.S.A.

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“…loss of activity (19), and saponin has been found to activate, a n d sodium taurocholate and Tween 20 to inhibit, the ACh-esterase of bovine erythrocytes (20). O n the other hand, Tween 20 has been used to extract ACh-esterase from h u m a n erythrocytes, with little loss in activity (21). It was found, however, that even rigorous homogenization of fraction 4A in 1 per cent sodium deoxycholate did not solubilize the ACh-esterase activity.…”

Section: Acetylcholinesterase In Electric Tissue Of Electrophorusmentioning

confidence: 99%

THE ASSOCIATION OF ACETYLCHOLINESTERASE AND MEMBRANE IN SUBCELLULAR FRACTIONS OF THE ELECTRIC TISSUE OF ELECTROPHORUS

Karlin

1965

The Journal of Cell Biology

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Subcellular fractions of the electric tissue of the main organ of the eel Electrophorus electricus were prepared in sucrose media by differential centrifugation and differential discontinuous gradient centrifugation. The distributions of acetylcholinesterase, cytochrome oxidase, DNA, and protein were determined. The appearance of the fractions was determined by phase contrast microscopy and by electron microscopy. A fraction prepared by differectial centrifugation at 30,000 g for 20 minutes in 0.89 M sucrose contained 63 per cent of the total acetylcholinesterase activity at 4 times the specific activity of that of the tissue homogenate. A subfraction prepared by centrifugation in a discontinuous density gradient showed a peak of total and relative specific acetylcholinesterase activity of 35 per cent and 1.9, respectively. The average over-all purification was 7 times. The acetylcholinesterase peak was below the cytochrome oxidase peak and above the DNA peak in the density gradient. The presence of acetylcholinesterase in the fractions was correlated with the presence of large fragments of the cell membrane; however, the presence of other tissue components was noted. The acetylcholinesterase associated with membrane was found to be activated by incubation with sodium deoxycholate. The possible use of the peak fraction containing membranes rich in acetylcholinesterase for the investigation of other components of the acetylcholine system and of other properties of the membrane is discussed.

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“…The extremely successful purification of electric eel cholinesterase by ROTHENBERG and NACHMANSOHN (1947) resulted in a preparation that hydrolysed acetylcholine at a rate of 400 mM/hr/mg protein. The specific activities of some other preparations were: 270 pM/hr/mg for ox red cell cholinesterase (COHEN and WARRINGA, 1953a), 440 pM/hr/mg for human red cell cholinesterase (ZITTLE et al, 1954), 360 pM/hr/mg for human serum cholinesterase (SURGENOR et al, 1949), and 12 mM/hr/mg for horse serum cholinesterase ( STRELITZ, 1944). Although insect cholinesterases attract much attention, especially with regard to their probable importance in organophosphate-poisoning and nerve transmission, the only successful attempt at purifying an insect cholinesterase was made by LORD (1961).…”

Section: Cholineanderasesmentioning

confidence: 99%

Partial purification and properties of flyhead cholinesterase

Dauterman¹,

Talens²,

Asperen³

1962

Journal of Insect Physiology

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Abstract-Houseflyhead cholinesterase was purified using the following steps: (1) freeze-drying of flyheads, (2) solubilization of the enzyme by butanol extraction, (3) ammonium sulphate precipitation at pH 7, (4) heat denaturation of proteins in the presence of acetylcholine for protection of the cholinesterase, (5) ammonium sulphate fractionation at pH 7 and at pH 6, (6) calcium phosphate gel absorption and elution, and (7) acetone fractionation.The final preparation, a solution in glass-distilled water, hydrolysed acetylcholine at a rate of 1600 pM/hr/mg of organic matter (157 x purification). It proved fairly stable and was used for studying some properties of the enzyme. The substrate specificity did not change much in the course of purification.The purified enzyme differed from purified bovine cholinesterase in that it hydrolysed butyrylcholine, triacetin, and phenylbutyrate at a much higher rate. The evidence strongly points to one single enzyme being responsible for the hydrolysis of all substrates studied, including butyrylcholine. Inhibition experiments with organophosphates indicate a probable turnover number in acetylcholine hydrolysis of about 100,000. Experiments on the influence of organic solvents showed that 2-3 y0 la-butanol increases the enzymic activity on choline esters about 60%) and that n-butanol, acetone, and ethanol all lower the rate of inhibition by an organophosphorus compound (diazoxon). Agar gel electrophoresis at pH 8.4 showed the cholinesterase to migrate, probably together with other proteins still present in the purified preparation, at a speed which is about 0.9 times the speed of human serum albumin.

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Purification of human red cell acetylcholinesterase

Cited by 43 publications

References 4 publications

Rapid and Complete Purification of Acetylcholinesterases of Electric Eel and Erythrocyte by Affinity Chromatography

Rapid and Complete Purification of Acetylcholinesterases of Electric Eel and Erythrocyte by Affinity Chromatography

THE ASSOCIATION OF ACETYLCHOLINESTERASE AND MEMBRANE IN SUBCELLULAR FRACTIONS OF THE ELECTRIC TISSUE OF ELECTROPHORUS

Partial purification and properties of flyhead cholinesterase

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