Marino Martinez-Carrion

Characterization of acetylcholine receptor isolated from Torpedo californica electroplax through the use of an easily removable detergent, β-d-octylglucopyranoside

Non-ionic detergents used for the solubilization and purification of acetylcholine receptor from Torpedo californica electroplax may remain tightly bound to this protein. The presence of detergent greatly hinders spectrophotometric and hydrodynamic studies of the receptor protein. beta-D-Octylglucopyranoside, however, is found to be effective in solubilizing the receptor from electroplax membranes with minimal interference in the characterization of the protein. The acetylcholine receptor purified from either octylglucopyranoside- or Triton X-100-solubilized extracts exhibits identical amino acid compositions, alpha-Bungarotoxin and (+)-tubocurarine binding parameters, and subunit distributions in SDS-polyacrylamide gels. The use of octylglucopyranoside allows for the assignment of a molar absorptivity for the purified receptor at 280 nm of approx. 530000 M-1 . cm-1. Additionally, successful reconstitution of octylglucopyranoside-extracted acetylcholine receptor into functional membrane vesicles has recently been achieved (Gonzales-Ros, J.M., Paraschos, A. and Martinez-Carrion, M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1796--1799). Removal of octylglucopyranoside by dialysis does not alter the specific toxin and antagonist binding ability of the receptor or its solubility at low protein concentrations. Sedimentation profiles of the purified acetylcholine receptor in sucrose density gradients reveal several components. Sedimentation coefficients obtained for the slowest sedimenting species agree with previously reported molecular weight values. Additionally, the different sedimenting forms exhibit distinctive behavior in isoelectric focusing gels. Our results suggest that both the concentration and type of detergent greatly influence the physicochemical behavior of the receptor protein.

Binding of anti-acetylcholine receptor antibodies inhibits the acetylcholine receptor mediated cation flux.

A fast kinetics, spectroscopic technique has been applied to the study of the transient cation flux associated to the binding of cholinergic agonist to native acetylcholine receptor (AcChR)-rich membrane vesicles in presence of anti-AcChR antibodies. The technique is based on the collisional quenching of an intravesicularly trapped fluorophore by externally added T1+ which substitutes for physiologically occurring cations. Presence of polyclonal Fab fragments from goat anti-AcChR antibodies bound to the membrane AcChR promotes a 80-90% inhibition on the observed rate constants of T1+ influx. The observed inhibition process appears to follow a non-competitive pattern between antibody and cholinergic ligand binding, suggesting that in the AcChR protein the antigenic sites responsible for ion translocation may be other than those involved in ligand binding.

Acetylcholine receptor from Torpedo. Preferential solubilization and efficient reintegration into lipid vesicles

The acetylcholine receptor has been effectively solubilized from Torpedo californica electroplax under defined conditions with the nonionic detergent, beta-D-octylglucopyranoside. Preferential solubilization of the receptor protein, with regard to yield and specific alpha-bungarotoxin binding activity, occurs in the absence of salt and diminishes when NaCl is present in the solubilization media (greater than or equal to 50 mM). Conversely, elevated salt concentrations increase the solubilization of bulk membrane proteins including the peripheral membrane enzyme, acetylcholine esterase. Additional selectivity for the solubilization of acetylcholine receptor can be obtained by adjusting the detergent to membrane phospholipid molar ratio within a narrow optimum range (4.1 to 6.7). Purified acetylcholine receptor and electroplax total lipid are utilized to reconstitute chemically excitable membrane vesicles. Reconstitution is achieved by dialysis of octylglucopyranoside from lipid/detergent/receptor protein mixed micelles and the resulting vesicles are analyzed by sucrose density gradient centrifugation. Extensive incorporation of the acetylcholine receptor within the lipid vesicles is obtained at lipid concentrations greater than 18 mg/ml with lipid/protein ratios ranging from 12/1 to 60/1 (w/w). Reconstituted receptor vesicles and native receptor-enriched membranes exhibit similar agonist-induced effluxes of 22Na+ with 50% of the maximum response occurring at carbamylcholine concentrations of 1.8 X 10(-5)M and 3.4 X 10(-5)M, respectively. At saturating carbamylcholine concentrations (10(-2)M) the agonist-induced efflux of 22Na+ for both native and reconstituted acetylcholine receptor is (6-7) X 10(13) cpm 22Na+ per mol of receptor. The efflux responses exhibited by either preparation can be effectively blocked by preincubation with carbamylcholine (desensitization). The similar behavior of native and reconstituted acetylcholine receptor indicates that octylglucopyranoside-purified receptor retains all of the necessary determinants for proper ligand binding and ion translocation.

A modification of α-bungarotoxin amino group residues with retention of binding properties to isolated and membrane-bound acetylcholine receptor☆

Abstract α-Bungarotoxin (α-Bgt), an α-neurotoxin, has been 14 C-methylated by treatment with [ 14 C]formaldehyde following NaCNBH 3 reduction. The methylation rate is fast (about 84% methylation in 15 min), with 12 methyl groups incorporated per mole of α-Bgt or a mean of 1.7 methyl groups per available amine residue. The specific activity of α-[ 14 C]Bgt is 768 mCi/mmol. Unlike most of the reported chemical modifications of α-neurotoxins, involving a high decrease of the toxin activity after modification, α-[ 14 C]Bgt retains 100% of its unmodified ability to bind to both isolated acetylcholine receptor (AcChR) and AcChR-enriched membrane fragments prepared from Torpedo californica . This lysyl residue modification does not perturb the toxin binding activity, probably, because the net positive charges of the ϵ-amino groups and amino-terminal residue remain unaltered. 14 C-Methylated α-Bgt appears better suited than 125 I-α-Bgt for use in AcChR binding studies because of the longer half-life of the isotope, and the apparent high uniformity of labeling of the toxin preparations.

Reductive methylation as a probe of the heat-labile α-bungarotoxin-acetylcholine receptor membrane complex: Evidence for surface interactions

alpha-Bungarotoxin (alpha-Bgt) is a potent postsynaptic neurotoxin which blocks neurotransmission by binding very tightly to the acetylcholine-receptor (AcChR) protein. We have previously shown (P. Calvo-Fernandez, and M. Martinez-Carrion (1981) Arch. Biochem. Biophys. 208, 154-159) that alpha-Bgt free in its native solution conformation incorporates 12 methyl groups when reductively methylated using formaldehyde and sodium cyanoborohydride. We now show that when the alpha-Bgt molecule is bound to the AcChR contained in native membranes prepared from Torpedo californica electroplax, the number of accessible methylation sites is significantly reduced. This favors a model of alpha-Bgt-AcChR interaction involving significant numbers of lysyl moieties distributed over a reasonably large surface of the toxin molecule. In addition, this paper presents a novel procedure for the rapid and nondestructive dissociation of the toxin-AcChR membrane complex which takes advantage of the thermal instability of the complex.

Substrate-mediated increased reactivity of a critical sulfhydryl group of crystals of cytoplasmic aspartate transaminase☆

Abstract The extramitochondrial isozyme of aspartate aminotransferase ( l -aspartate:2-oxoglutarate aminotransferase EC 2.6.1.1) contains a cysteinyl residue (cysteine-390) which, in the presence of substrate, displays enhanced reactivity toward sulfhydryl reagents. To gain insight into the structural similarity of the enzyme in solution compared to its crystalline state and into the type of structural change induced by substrates, the reactivity of Cys-390 in the crystalline enzyme has been studied. The flat yellow plates, crystallized from polyethylene glycol, form spectroscopically detectable enzyme-substrate complexes (C. M. Metzler, D. E. Metzler, D. S. Martin, R. Newman, A. Arnone, and P. Rodgers, 1978, J. Biol. Chem. 253, 5251–5254). The crystals, both in the presence and absence of the substrate pair, glutamate and α-ketoglutarate, were treated with N-ethylmaleimide or N-ethyl[1-14C]maleimide and the extent of the reaction was monitored by the colorimetric sulfhydryl reaction with 5,5′-dithiobis-2-nitrobenzoic acid, by amino acid analysis, by radioactivity incorporated, and by the measurement of enzyme activity. A cysteine residue was modified only in the presence of substrate; the crystals remained undamaged. Since, any large conformational change in the enzyme would either be prevented by the crystalline lattice or would disrupt its integrity, it is concluded that the enhanced reactivity of cysteine-390 in the presence of substrates must be due to only a small local conformational change in the substrate binding region.

Absorption filtration. A tool for the measurement of ion tracer flux in native membranes and reconstituted lipid vesicles

A rapid, simple and reproducible method has been developed for the measurement of ion tracer flux with both native membrane vesicles and reconstituted lipid vesicle systems. Following the absorption of vesicles onto glass fiber filters, tracer flux is performed directly upon the deposited samples. In contrast to the more conventional vacuum and gel filtration techniques, absorption filtration exhibits comprehensive data retrieval whereby the removal of extravesicular ions, the retention of intravesicular ions and the amount of ions fluxed can be accurately analyzed. Both influx and efflux assays have been designed to measure the carbamylcholine-induced flux of 22Na+ which is characteristic of acetylcholine receptor-enriched membranes from Torpedo californica electroplax. The flux signal-to-background noise ratio is maximized in the efflux assay, since agonist activation is performed subsequent to the exhaustive removal of extravesicular tracer. An interesting feature of the influx assay is that the agonist-induced uptake of 22Na+ can be repeated with the original vesicles which additionally maximizes the flux signal. With either approach, the inactivation of ionophoric activity due to prolonged exposure to agonists (desensitization) can be reversed upon removal of agonist without dilution of the deposited samples. Due to the large array of glass fiber filters and ion-exchange disks, the absorption filtration technique should be able to accommodate the transport and binding of soluble molecules to a variety of intact cells, membranes and reconstituted lipid vesicles.

Effect of anti-acetylcholine receptor Fab fragments from goats on functional membrane-bound receptor

Abstract Fab fragments prepared from goats displaying experimental autoimmune myasthenia gravis (EAMG) recognize and bind to membrane-bound acetylcholine receptor (AcChR) from Torpedo californica electroplax. Presence of bound Fab fragments leads to an alteration in a number of functional properties associated with the AcChR-rich membranes. Titration of membrane-bound AcChR with (EAMG)Fab fragments reveals a maximum of three to four high-affinity Fab fragments which cannot be displaced by α-bungarotoxin (α-Bgt). Presence of bound Fab fragments on the membranes does not alter the affinity of the AcChR for ligands tested (propidium, d-tubocurarine, decamethonium) but reduces the total amount of ligand capable of being bound. Preincubation with (EAMG)Fab, previously shown to slow the time-dependent binding of α-Bgt to solubilized AcChR, has now been shown to exert a similar effect on membrane-bound forms of the AcChR; only high-affinity Fab fragments are necessary to cause this effect. Presence of bound (EAMG)Fab seems to highly diminish but not eliminate the agonist induced “sensitization-desensitization” phenomenon characteristic of membrane-bond AcChR. These same bound Fab fragments do not appear to alter the agonist-induced transfer of 22Na+ across AcChR-enriched membranes at carbamylcholine concentrations sufficient to elicit a 100% flux response. Conversely, the presence of bound Fab shifts the dose-response curves to lower carbamylcholine concentrations further indicating a decrease in the magnitude of the desensitization. These results suggest interference by bound anti-AcChR antibodies on AcChR function in our “in vitro-in situ” myasthenic membrane model, which might therefore be useful in the study of primary membrane events in the impaired neuromuscular transmission seen in the human disease myasthenia gravis.

Rapid purification of a phospholipase-free α-bungarotoxin: Maintenance of cation barriers of acetylcholine receptor membranes upon preincubation with purified toxin

The purification of highly homogeneous, phospholipase-free alpha-bungarotoxin (alpha-Bgt) from the venom of the elapid Bungarus multicinctus or from commercial samples of alpha-Bgt is described. The method combines a conventional procedure for the purification of alpha-Bgt [D. Mebs, K. Narita, S. Iwanaga, Y. Samejima, and C. Y. Lee (1972) Hoppe-Seylers Z. Physiol. Chem. 353, 243-262] with high-resolution gel-filtration and cation-exchange chromatography steps to remove membrane-damaging, contaminating phospholipase activity. The procedure also removes contaminating radioactive peptides from commercial preparations of 125I-alpha-Bgt. Apparent homogeneity of the purified alpha-Bgt (referred to as fraction D in the text), as well as the absence of contaminating phospholipase A2 activity, is assessed by (i) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, (ii) gel-filtration and cation-exchange high-performance liquid chromatography, (iii) direct measurements of phospholipase A2 activity under conditions where very low enzymatic levels should be detected, (iv) lack of interference with the passive cation permeability properties of acetylcholine receptor membranes, (v) competitive inhibition of 125I-alpha-Bgt binding to the acetylcholine receptor membranes, and (vi) amino acid analysis and end-group (C- and N-terminus) determination. alpha-Bgt preparations subjected to these criteria do not exert the increase in membrane passive permeability to cations detected with other laboratory or commercial samples of alpha-Bgt. Availability of the new alpha-Bgt preparation allows for an assessment of the inertness of alpha-Bgt on lipid membrane properties while preventing cholinergic ligand binding to nicotinic acetylcholine receptor-rich membranes. These conditions are necessary for experiments requiring maintenance of the physical and phospholipid integrity of membranes.

Pyrenesulfonyl azide: A covalent probe permitting invitro desensitization of labeled acetylcholine-rich membrane fragments from Torpedocalifornica

Abstract Acetylcholine receptor-rich membrane fragments from Torpedo californica electroplax after covalent labelling at the protein-lipid boundary by nitrenes generated in situ from pyrenesulfonyl azide can bind [ 125 I]-α-bungarotoxin . The covalent attachment of 6–8 molecules of the fluorescent probe/receptor molecule also does not perturb the marked effect on the rate of α-bungarotoxin binding to electroplax membranes exerted by their preincubation with carbamylcholine. This phenomenon, which is analogous to pharmacological desensitization of receptors in synaptic junctions, is fully reversible upon removal of carbamylcholine (Quast, V., Schmerlik, M., Lee, T., Witzemann, V., Blanchard, V. and Raftery, M.A. (1978) Biochemistry 17 , 2405–2414). Torpedo electroplax membranes, whether tagged with the covalent probe or freshly isolated, regain the original fast rate of α-bungarotoxin binding upon dilution of carbamylcholine.