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Biomedical subjects

A Karlin

Publications and source records attributed to A Karlin.

At least 73 records · Page 4Linked to original sources
PubMed1979

Activation of the frog sartorius acetylcholine receptor by a covalently attached group.

The frog sartorius motor endplate was treated with the specific disulfide bond reducing agent dithiothreitol and subsequently exposed to a covalently reacting compound (the nitrophenyl ester of p-carboxyphenyltrimethylammonium iodide, NPTMB) known to activate the dithiothreitol-reduced acetylcholine receptor in Electrophorus electroplax. NPTMB causes a maximum depolarization of about 35 mV when applied to the dithiothreitol-treated sartorious motor endplate. It is ineffective on postjunctional membrane prior to disulfide bond reduction and on extrajunctional regions, reduced or unreduced. High concentrations of a competitive antagonist such as (+)-tubocurarine prevent reaction between NPTMB and the reduced receptor and cause a repolarization of the membrane when applied to the already-depolarized preparation. We conclude that in frog muscle, as in electroplax, the attached activator bridges the acetylcholine binding site of the reduced receptor between a sulfhydryl group, to which it is covalently bound, and a negative subsite, with which it forms a reversible ionic band.

Acetylcholine↗
PubMed1979

Voltage fluctuations at the frog sartorius motor endplate produced by a covalently attached activator.

The depolarization that develops after covalent attachment of trimethylammonium benzoyl to the dithiothreitol-reduced frog sartorius acetylcholine receptor is accompanied by a small increase in voltage fluctuations. The amplitude of the elementary voltage event produced by the covalently attached activator is about 0.04 microV, almost an order of magnitude below the acetylcholine shot-effect amplitude in the control preparation, and about one-fourth the acetylcholine shot amplitude after disulfide-bond reduction. Spectral density plots of trimethylammonium-benzoyl noise can be analyzed in terms of two relaxation rates that bracket the single rate observed in response to acetylcholine.

Acetylcholine↗
PubMed1979

An analysis by low-angle neutron scattering of the structure of the acetylcholine receptor from Torpedo californica in detergent solution.

The acetylcholine receptor from the electric tissue of Torpedo californica is a large, integral membrane protein containing four different types of polypeptide chains. The structure of the purified receptor in detergent solution has previously been investigated by sedimentation analysis and gel filtration. Sedimentation analysis yielded a molecular weight of 250,000 for the protein moiety of the receptor monomer-detergent complex; hydrodynamic characteristics such as the Stokes radius, however, refer to the receptor-detergent complex. In this paper we report the results of our use of low-angle neutron scattering to investigate the shape of the receptor-detergent (Triton X-100 from Rohm & Haas Co., Philadelphia, Pa.) complex and separately of its protein and detergent moieties. By adjustment of the neutron-scattering density of the solvent with D2O to match that of one or the other of the moieties, its contribution to the scattering can be nearly, if not completely, eliminated. Neutron scattering from Triton X-100 micelles established that this detergent is contrast matched in approximately 18% D2O. Scattering measurements on the receptor-detergent complex in this solvent yielded a radius of gyration of the acetylcholine receptor monomer of 46 +/- 1A. The radius of gyration and molecular volume (305,000 A3) of the receptor are inconsistent with a compact spherical shape. These parameters are consistent with, for example, a prolate cylinder of dimensions (length x diameter) approximately 150 x approximately 50 A or an oblate cylinder, approximately 25 x approximately 130 A. More complex shapes are possible and in fact seem to be required to reconcile the present results with previous electron microscopic and x-ray analyses of receptor in membrane and with considerations of the function of the receptor in controlling ion permeability. The neutron-scattering data yield, in addition, an independent determination of the molecular weight of the receptor protein (240,000 +/- 40,000), the extent of Triton X-100 binding in the complex (approximately 0.4 g/g protein), and from the extended scattering curve, an approximation to the shape of the receptor-Triton X-100 complex, namely an oblate ellipsoid of axial ratio 1:4.

Animals↗
PubMed1978

Molecular weight in detergent solution of acetylcholine receptor from Torpedo californica.

Acetylcholine receptor extracted in detergent solution from the electric tissue of Torpedo californica and purified by affinity chromatography contains predominantly two molecular weight species. These have been separated by sedimentation in a sucrose density gradient, and their molecular properties have been determined by sedimentation equilibrium and sedimentation velocity measurements in the analytical ultracentrifuge. The molecular weights of these species have been determined, without prior determination of the extent of detergent bound to them, by the adjustment of solvent density with D2O so as to blank out the contribution of bound detergent to the sedimentation potential. The molecular weights of the protein moieties are 250 000 and 500 000. Since these species are identical in specific activity and polypeptide composition they are related as monomer and dimer. The hydrodynamic properties of the detergent complexes of monomer and dimer were derived from combined measurements of sedimentation equilibrium and sedimentation velocity. The S20,w'S are 8.6 S and 12.8 S and the Stokes radii are 7.3 nm and 9.5 nm. For both monomer and dimer, the ratio of the Stokes radius to the minimum possible radius for the protein-detergent complex falls outside the range of values for globular proteins.

Acetylcholine↗
PubMed1978

Binding of antibodies to acetylcholine receptors in Electrophorus and Torpedo electroplax membranes.

Antisera against purified acetylcholine receptors from the electric tissues of Torpedo californica and of Electrophorus electricus were raised in rabbits. The antisera contain antibodies which bind to both autologous and heterologous receptors in solution as shown by an immunoprecipitation assay. Antibodies in both types of antisera bind specifically to the postjunctional membrane on the innervated surface of the intact electroplax from Electrophorus electric tissue as demonstrated by an indirect immunohistochemical procedure using horseradish peroxidase conjugated to anti-rabbit IgG. Only anti-Electrophorus receptor antisera, however, cause inhibition of the receptor-mediated depolarization of the intact Electrophorus electroplax. The lack of inhibition by anti-Torpedo receptor antibodies, which do bind, suggests that the receptor does not undergo extensive movement during activity. The binding of anti-Torpedo antibodies to receptor-rich vesicles prepared by subcellular fractionation of Torpedo electric tissue was demonstrated by both direct and indirect immunohistochemical methods using ferritin conjugates. These vesicles can be conveniently collected and prepared for electron microscopy on Millipore filters, a procedure requiring only 25 micrograms of membrane protein per filter. In addition, it was possible to visualize the binding of anti-Torpedo receptor antibodies directly, without ferritin. These anti-Torpedo receptor antibodies, however, do not inhibit the binding of acetylcholine or of alpha-neurotoxin to receptor in Torpedo microsacs but do inhibit binding of alpha-neurotoxin to Torpedo receptor in Triton X-100 solution. It is likely that the principal antigenic determinants on receptor are at sites other than the acetylcholine-binding sites and that inhibition of receptor function, when it occurs, may be due to a stabilization by antibody binding of an inactive conformational state.

Acetylcholine↗
PubMed1977

Affinity alkylation labels two subunits of the reduced acetylcholine receptor from mammalian muscle.

The acetylcholine receptor from denervated rat skeletal muscle was purified by affinity chromatography and, after reduction, was treated with the affinity alkylating agent 4-(N-maleimido)benzyltri[3H]methylammonium iodide. The receptor specifically incorporated approximately 1 mol of alkylating agent per mol of 125I-labeled alpha-bungarotoxin bound. Analysis of the labeled receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that two subunits were labeled; their apparent molecular weights were 45,000 and 49,000. These results suggest that the affinity reagent labels a second site for acetylcholine binding in the muscle receptor that is not labeled in receptors from Electrophorus or Torpedo.

Acetylcholine↗