THE ASSOCIATION OF ACETYLCHOLINESTERASE AND MEMBRANE IN SUBCELLULAR FRACTIONS OF THE ELECTRIC TISSUE OF <i>ELECTROPHORUS </i>

Karlin, Arthur

doi:10.1083/jcb.25.2.159

Cited by 49 publications

(8 citation statements)

References 19 publications

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“…A subcellular fraction rich in membrane-bound acetylcholinesterase (M\-AChE) has been prepared from electric tissue of Electrophorus electricus. 2 The acetylcholinesterase activity of this fraction shows an anomalous dependence on pH, similar to that described for a model system in which papain is bound to a collodion membrane.4 In both cases, this effect can be explained as due to local pH changes in the vicinity of the membrane-bound enzyme consequent to the hydrolysis of substrate.…”

supporting

confidence: 53%

Effect of local pH changes caused by substrate hydrolysis on the activity of membrane-bound acetylcholinesterase.

Silman¹,

Karlin²

1967

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

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supporting

confidence: 53%

Effect of local pH changes caused by substrate hydrolysis on the activity of membrane-bound acetylcholinesterase.

Silman¹,

Karlin²

1967

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

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“…The evidence is based on electron microscopy combined with histochemical studies, and on subcellular fractionation (12)(13)(14). However, the exact relationship of the enzyme to the membrane has not been clarified (15,16).…”

Section: Discussionmentioning

confidence: 99%

Molecular Structures of Acetylcholinesterase from Electric Organ Tissue of the Electric Eel

Dudai

Herzberg

Silman

1973

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

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Three purified molecular forms of acetylcholinesterase (EC 3.1.1.7) with sedimentation coefficients of 18 S, 14 S, and 11 S were studied by analytical ultracentrifugation and electron microscopy. The three species have molecular weights of (1.1 ± 0.1) X 106, (7.5 4 1.5) X 106, and (3.35 4-0.25) X 106, respectively. Electron micrographs reveal that the 18S and 14S forms are asymmetric, composed of a head, containing a large number of subunits, and an elongated tail. The 11 S form of acetylcholinesterase is apparently a tetrameric structure devoid of the tail. Maleylation of 18S and 14S acetylcholinesterases abolishes their tendency to aggregate at low ionic strength.Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) in extracts of fresh electric organ tissue of electric eels consists of several components distinguished by their sedimentation coefficients (1, 2). The major component is an 18S form, which aggregates at low ionic strength (2, 3). A 14S component, which also aggregates at low ionic strength, is present in smaller amounts. Treatment of electric organ tissue with proteolytic enzymes or autolysis converts all the acetylcholinesterase to an 11S form, which does not aggregate at low ionic strength and is not present in fresh tissue (2, 3).We have purified, by affinity chromatography, the different molecular forms of acetylcholinesterase present in fresh tissue or obtained after proteolysis (3, 4). Purified 18S and 14S acetylcholinesterases retain their tendency to aggregate at low ionic strength, and the 18S, 14S, and 11S acetylcholinesterases all display similar patterns on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol (4). Massouli6 et al.(5) have suggested, on the basis of gel-filtration studies, that 18S and 14S acetylcholinesterases have an asymmetric structure, in contrast with 11S acetylcholinesterase, which is globular.In the following we will show, by electron microscopy and ultracentrifugation, that 18S and 14S acetylcholinesterases differ markedly in their quaternary structure from the 11S form. Similar electron microscopic observations have been recently reported by Rieger et al. (6). We will also show that the tendency of 18S and 14S acetylcholinesterases to aggregate at low ionic strength can be prevented by chemical modification. The relevance of these observations to the relationship of acetylcholinesterase with the electroplax membrane will be discussed. METHODS 18S, 14S, and llS forms of acetylcholinesterase were purified by affinity chromatography (3, 4). The 18S and -14S enzymes were separated from each other by sucrose gradient centrifugation as follows: aliquots of 1.8 ml of a mixture of purified 18S and 14S enzymes (containing about 1 mg/ml of enzyme) were layered on a 5-20% linear sucrose gradient in 1.0 M NaCl-0.01 M phosphate (pH 7.0) of total volume of 28 ml, with a 5-ml cushion of 60% sucrose in the same buffer at the bottom. Centrifugation was performed in an SW27 rotor in an L2 65-B Beckman preparative ultra...

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“…This fraction is referred to as "purified membranes." In contrast to the crude preparation, it is largely free from both connective fibers and the nucleated microcells that spontaneously form during homogenization of this tissue (Karlin, 1965).…”

Section: Membrane Preparationsmentioning

confidence: 99%

Thiamine Triphosphatase in the Membranes of the Main Electric Organ of Electrophorus electricus: Substrate‐Enzyme Interactions

Bettendorff

Grandfils

Wins

et al. 1989

Journal of Neurochemistry

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The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate (TTP). Membrane fractions prepared from this tissue contain a thiamine triphosphatase that is strongly activated by anions and irreversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion transport inhibitor. Kinetic parameters of the enzyme are markedly affected by the conditions of enzyme preparation: In crude membranes, the apparent Km is 1.8 mM and the pH optimum is 6.8, but trypsin treatment of these membranes or their purification on a sucrose gradient decreases both the apparent Km (to 0.2 mM) and the pH optimum (to 5.0). Anions such as NO3- (250 mM) have the opposite effect, i.e., even in purified membranes, the pH optimum is now 7.8 and the Km is 1.1 mM; at pH 7.8, NO3- increases the Vmax 24-fold. TTP protects against inhibition by DIDS, and the KD for TTP could be estimated to be 0.25 mM, a value close to the apparent Km measured in the same purified membrane preparation. Thiamine pyrophosphate (0.1 mM) did not protect against DIDS inhibition. At lower (10(-5)-10(-6) M) substrate concentrations, Lineweaver-Burk plots of thiamine triphosphatase activity markedly deviate from linearity, with the curve being concave downward. This suggests either anticooperative binding or the existence of binding sites with different affinities for TTP. The latter possibility is supported by binding data obtained using [gamma-32P]TTP. Our data suggest the existence of a high-affinity binding site (KD of approximately 0.5 microM) for the Mg-TTP complex.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Cited by 49 publications

References 19 publications

Effect of local pH changes caused by substrate hydrolysis on the activity of membrane-bound acetylcholinesterase.

Effect of local pH changes caused by substrate hydrolysis on the activity of membrane-bound acetylcholinesterase.

Molecular Structures of Acetylcholinesterase from Electric Organ Tissue of the Electric Eel

Thiamine Triphosphatase in the Membranes of the Main Electric Organ of Electrophorus electricus: Substrate‐Enzyme Interactions

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