Separation of Multiple Molecular Forms of Acetylcholinesterase by ...

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Department of Pharmacology, University of Oxford, South Parks Road, Oxford. OX1 3QT, U.K.. Rabbit brain contains five multiple molecular forms of soluble ...
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Separation of Multiple Molecular Forms of Acetylcholinesterase by Using Affinity Chromatography:Isolation of the ‘Secretory’ Form ANTHONY J. HODGSON, IAN W. CHUBB and A. DAVID S M l T H Department of Pharmacology, University of Oxford, South Parks Road, Oxford OX1 3QT, U . K . Rabbit brain contains five multiple molecular forms of soluble acetylcholinesterase (EC 3.1.1.7). Only one of these forms is found in cerebrospinal fluid; this form of acetylcholinesterase is named ‘secretory’ acetylcholinesterase. Electrical stimulation of the peripheral o r central nervous system increases the concentration of the secretory form in cerebrospinal fluid (Chubb et a/., 1976; Greenfield & Smith, 1976). Edrophonium chloride was coupled to epoxy-activated Sepharose according to the manufacturer’s (Pharmacia) recommendations. Soluble brain extracts were prepared by homogenizing whole brains to a final concentration of 10% (w/v) in 0.3~-sucroseor in 5m~-Tris/HC1/83mM-glycine, pH 8.2, containing 0.15 M-NaCt. The homogenates were frozen and thawed twice before centrifugation at IOOOOOg,,. for 60min. Multiple molecular forms of the enzyme were detected by electrophoresis (Clarke, 1964), followed by histochemical staining for enzyme activity (Coupland & Holmes, 1957). Protein was detected by staining with Coomassie Brilliant Blue G250 (Reisner at al., 1975). Thesolublefractionfromrabbit braincontained 12%ofthetotal activityin the homogenate. Four molecular forms were observed on polyacrylamide gels by histochemistry. One of these forms corresponded to that found in cerebrospinal fluid. A portion (20ml) of the homogenate was applied to the column (volume 10ml) at a flow rate of I2 ml/h, and 37% of the enzyme and 90% of the protein passed through the column unretarded. Electrophoretic analysis revealed that only the non-secretory forms were present in this unbound fraction. Washing the column with several column volumes of 5Om~-sodium phosphate/potassium phosphate buffer, pH 7.0, removed traces of enzyme and a further 3 % of the protein. Increasing the salt concentration t o 0 . 5 in ~ NaCl eluted I 1 ‘4 of the enzyme applied and all the remaining protein (recovery 105%). The column was then eluted with 1OmM-edrophonium chloride in the phosphate buffer. Fractions (2nd) were collected and analysed electrophoretically. Those fractions containing histochemically detectable enzyme were pooled and dialysed exhaustively against phosphate buffer; 28 % of the enzyme activity was recovered. Electrophoresis showed that this consisted of only one form, corresponding t o that found in cerebrospinal fluid. The protein concentration in the eluate was not measurable by the Lowry ef a/. (1951) method. When unbound enzyme was reapplied to the column no binding occurred. Furthermore the isolated secretory form of enzyme was stable at 4°C for several weeks, showing n o tendency to aggregate o r dissociate into other molecular forms. Similar experiments were done with guinea-pig and mouse brain, which contain three and four soluble forms of acetylcholinesterase respectively. I n both cases the only form of the enzyme bound by the column was the one also present in cerebrospinal fluid. Guinea-pig serum contains more than 10 times as much cholinesterase (EC 3. I . I .8) as acetylcholinesterase activity. When guinea-pig serum was applied to the column none of the cholinesterase bound. The acetylcholinesterase could be eluted free of cholinesterase by 10mM-edrophonium. Electrophoresis showed that only one molecular form was present in the eluate. A similar experiment was carried out with the same result with mouse serum, which contains an even greater excess of cholinesterase. Bovine adrenal medulla contains five forms of soluble acetylcholinesterase (Chubb & Smith, 1975a). Two-dimensional polyacrylamide-gel electrophoresis on 6 % gels in the first dimension and into a polyacrylamide gradient (4-24%) as the second dimension showed that the secretory form of enzyme (Chubb & Smith, 19756), has the greater molecular size. This form was the only one bound by the affinity gel. The present results illustrate that edrophonium coupled to agarose is able to bind selectively only one of the several forms of soluble acetylcholinesterase. The exact basis of this selectivity is unknown but it is not a result of differences in accessibility to the 1978

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ligand because the smaller molecular forms, which would be better able to penetrate the agarose gel, are not bound, whereas the larger form does bind. The gel can also discriminate between acetylcholinesterase and cholinesterase. This is unexpected because edrophonium is a competitive inhibitor of both enzymes. This suggests that the coupling of a side chain to the meta-hydroxy group of edrophonium alters the inhibitory properties of the molecule. Chubb, I. W. & Smith, A. D. (19757) Proc. R . Soc. London Ser. B 191,245-261 Chubb, I. W. &Smith, A. D. (19756) Proc. R. SOC.London Ser. B 191,263-269 Chubb, I. W., Goodman, S. &Smith, A. D. (1976) Neuroscience 1,57-62 Clarke, J. T. (1964) Ann. N. Y . Acud. Sci. 121,428-436 Coupland, R. E. & Holmes, R. L. (1957) Q. J. Microsc. Sci. 98,327-330 Greenfield, S. A. &Smith, A. D. (1976)J. Physiol. (London) 258, 108P-109P Lowry, 0. H., Rosebrough, N. J., Farr,A. L. &Randall, R. J. (1951)J. Biol. Chem. 193,265-275 Reisner, A. H., Nemes, P. & Bucholtz, C. (1975) Anal. Biochem. 64, 509-516

Size and Molecular Properties of the Acetylcholine-Receptor Protein of Muscle E R I C A. BARNARD,* J. OLIVER DOLLY,* MATTHEW LO* and TIMOTHY MANTLE? *Department of Biochemistry, fmperiaf College, London S W7 2AZ, U.K.,

and ?Trinity College, Dublin 2, Ireland The nicotinic acetylcholine receptor has been isolated as a protein from fish electric organs in a number of laboratories, and also from denervated mammalian muscle (Dolly & Barnard, 1975, 1977). When purified from muscle, the acetylcholine-recognition unit of the receptor has been shown to contain essentially a single type of subunit, a glycoprotein of 41 000mol.wt. (Shorr e t a / . , 1978). From electroplaques, most investigators have reported two to four types of subunit in the pure protein (Claudio & Raftery, 1977; Hucho et a / . , 1976; and other studies reviewed by Karlin et al., 1975), but Sobel et a / . (1977) have reported very largely one subunit of 40000mol.wt from Torpedo. We report here on the size of the oligomeric mammalian muscle receptor both as a pure protein and in its original membrane-bound state. Muscles, denervated 3-4 weeks, were removed as noted from cats, rabbits or rats. Labelling of the receptor, and its assay (Dolly & Barnard, 1974), used its specific reaction with [3H]a-neurotoxin (Barnard et a/., 1971), in this case an a-neurotoxin from Naja melanoleuca venom, labelled at 5.7-1 8 Ci/mmol by catalytic halogen-exchange tritiation at a single histidine carbon (A. Menez, E. A. Barnard, J . 0. Dolly & P. Fromageot, unpublished work). Purification of the free receptor involved extraction in Triton X-100 media, affinity chromatography on an a-neurotoxin-Sepharose column, biospecific elution with carbamoylcholine and chromatography on DEAE-Sepharose, with details as noted by Dolly et a / . (1977). Specific activity (in specific toxin-binding sites per g of protein) was (from cat or rabbit leg muscles) at this stage 3000-6000nmoI/g. A repeat cycle of the affinity chromatography on these preparations (from cat) always increased the specific activity, up to a maximum of l0500nmol/g. The active receptor was homogeneous in gel isoelectric focusing, with pl5.0. It bound nicotinic cholinergic ligands with the relatively high affinities observed (Barnard et a/., 1977) on membrane fragments from denervated muscle, and all the [3H]toxin binding was suppressed by O.lmMacetylcholine or (+)-tubocurarine. The rate constant for reaction of the receptor (at 2 5 T , pH8, in 0.1 % Triton X-100) with Naja melanoleuca [3H]a-neurotoxin was 2 . 9 ~1 0 5 M - 1 . ~ 1 . Receptor preparations were analysed by sucrose-density-gradient centrifugation, with the results shown in Table 1. After only a brief extraction from muscle at 4"C, and

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