Search PubMed⌕ Search

Biomedical subjects

J Lindstrom

Publications and source records attributed to J Lindstrom.

At least 181 records · Page 10Linked to original sources
PubMed1980

Purification of acetylcholine receptors, reconstitution into lipid vesicles, and study of agonist-induced cation channel regulation.

We report the purification of acetylcholine receptors with active agonist-regulated cation channels from Torpedo californica electric organ tissue by five methods. In one method, previously used by others, contaminating proteins were removed from partially purified membranes by alkaline extraction, preserving membrane integrity throughout the procedure. In the other four methods, acetylcholine receptors were purified after solubalization with sodium cholate. The continual presence of soybean lipid in mixed micelles with cholate was required to prevent irreversible inactivation of the cation channel. Solubilized receptors were purified by affinity chromatography using either Naga naja siamensis toxin III or concanavalin A coupled to agarose. Sucrose gradient centrifugation was also used to purify solubilized receptors. The best method combined affinity chromatography on toxin-agarose and concanavalin A agarose. Receptors purified by all five methods were incorporated into soybean lipid vesicles by the cholate dialysis technique. The agonist-regulated cation channels of the receptors were equally active after reconstitution, independent of the method used for purification. All reconstituted vesicle preparations were similar in preferential orientation of acetylcholine receptor toward the external surface, dose-response to carbamylcholine, carbamylcholine-induced desensitization, and carbamycholine-induced influx of 22Na+ per mol of receptor. Carbamylcholine-induced 22Na+ influx/receptor was greater after reconstitution than in native vesicles. This was because, in native vesicles, carbamylcholine-induced 22Na+ influx was limited by equilibration of the internal volume of the vesicles with the external 22Na+ concentration, whereas in reconstituted vesicles 22Na+ influx was limited by desensitization of the receptor molecule. We demonstrate that only one of the two toxin binding sites on the receptor monomer, the one which can be affinity alkylated with 4-(N-maleimido)benzyltrimethylammonium, controls the carbamylcholine-induced opening of the cation channel.

Acetylcholine↗
PubMed1980

Acetylcholine receptors from Torpedo and Electrophorus have similar subunit structures.

Previously, acetylcholine receptor purified from the electric organs of electric eels (Electrophorus electricus) and electric rays (Torpedo californica) (torpedo) had appeared to differ in subunit structure. Receptor from torpedo has the subunit structure alpha 2 beta gamma delta, but subunits corresponding only to alpha, beta, and gamma had been observed in receptor from eel. Here we report that if membrane fragments of eel electric organ are prepared and detergent extracted in the presence of iodoacetamide, then receptor purified from the extract contains a fourth subunit. Using monoclonal antibodies as well as conventional antisera, we show that the newly recognized subunit of receptor from eel corresponds to the delta subunit of torpedo. A monoclonal antibody to the delta subunit of torpedo cross-reacts with the gamma subunit and shows a similar cross-reaction between the delta' and gamma' subunits of receptor from eel, indicating the presence of an unexpected structural similarity. Although the function of the beta, gamma, and delta subunits remains unknown, these results support the concept that receptors from the electric organs of several species and probably also from muscle share a similarly complex subunit structure.

Acetylcholine↗
PubMed1980

Reconstitution of purified acetylcholine receptors with functional ion channels in planar lipid bilayers.

Acetylcholine receptor, solubilized and purified from Torpedo californica electric organ under conditions that preserve the activity of its ion channel, was reconstituted into vesicles of soybean lipid by the cholate-dialysis technique. The reconstituted vesicles were then spread into monolayers at an air-water interface and planar bilayers were subsequently formed by apposition of two monolayers. Addition of carbamoylcholine caused an increase in membrane conductance that was transient and relaxed spontaneously to the base level (i.e., became desensitized). The response to carbamoylcholine was dose dependent and competitively inhibited by curare. Fluctuations of membrane conductance corresponding to the opening and closing of receptor channels were observed. Fluctuation analysis indicated a single-channel conductance of 16 +/- 3 pS (in 0.1 M NaCl) with a mean channel open time estimated to be 35 +/- 5 ms. Thus, purified acetylcholine receptor reconstituted into lipid bilayers exhibited the pharmacological specificity, activation, and desensitization properties expected of this receptor in native membranes.

Animals↗
PubMed1980

Functional equivalence of monomeric and dimeric forms of purified acetylcholine receptors from Torpedo californica in reconstituted lipid vesicles.

Acetylcholine receptors from Torpedo californica electric organ were solubilized and purified under conditions which prevent inactivation of the agonist-regulated cation channels. The dimer form of the receptors was preserved during purification. Treatment with reducing agents converted dimers into monomers. Receptor monomers and dimers were separately reconstituted into soybean lipid vesicles by the cholate dialysis technique. Reconstituted monomers and dimers were functionally equivalent with respect to their carbamylcholine-induced dose-dependent uptake of 22Na+, the total flux of 22Na+ per receptor during the permeability response, and the occurrence of desensitization. Evidence against non-covalent association of monomers to produce dimeric functional units was obtained using glutaraldehyde as a crosslinking agent. These results show that both the acetylcholine-binding sites and the agonist-regulated cation-specific channel are contained within the alpha 2 beta gamma delta subunit structure of the acetylcholine receptor monomer.

Acetylcholine↗
PubMed1980

Experimental autoimmune myasthenia gravis.

Injection of animals with purified acetylcholine receptor in complete Freund's adjuvant causes development of antibodies which crossreact with receptors in muscle. The crossreacting antibodies impair neuromuscular transmission. Animals with experimental autoimmune myasthenia gravis (EAMG) are excellent models for studying the complex mechanisms by which the autoimmune response to receptor in myasthenia gravis causes muscle weakness. This review first briefly describes the discovery of EAMG. Then, to provide the necessary perspective, receptor structure and function and properties of anti-receptor antibodies are discussed, followed by a brief review of the pathological mechanisms in EAMG.

Acetylcholine↗
PubMed1979

Biochemical properties of acteylcholine receptor subunits from Torpedo californica.

Four polypeptide chains composing acetylcholine receptors from the electric organ of Torpedo californica were purified by preparative electrophoresis in sodium dodecyl sulfate. Their apparent mole ratio alpha/beta/gamma/delta is 2:1:1:1. These chains are not readily distinguished by amino acid or carbohydrate composition but are distinguished by apparent molecular weight and polypeptide maps. By peptide maps, no extensive homology is evident between these chains or between any of these chains and higher molecular weight chains found in receptor-enriched membrane fragments.

Acetylcholine↗
PubMed1979

Immunochemical similarities between subunits of acetylcholine receptors from Torpedo, Electrophorus, and mammalian muscle.

Polypeptide chains composing acetylcholine receptors from the electric organs of Torpedo californica and Electrophorus electricus were purified and labeled with 125I. Immunochemical studies with these labeled chains showed that receptor from Electrophorus is composed of three chains corresponding to the alpha, beta, and gamma chains of receptor from Torpedo but lacks a chain corresponding to the delta chain of Torpedo. Experiments suggest that receptor from mammalian muscle contains four groups of antigenic determinants corresponding to all four of the Torpedo chains. Binding of 125I-labeled chains was measured by quantitative immune precipitation and electrophoresis. Antisera to the following immunogens were used: denatured alpha, beta, gamma, and delta chains of Torpedo receptor, native receptor from Torpedo and Electrophorus electric organs and from rat and fetal calf muscle, and human muscle receptor (from autoantisera of patients with myasthenia gravis). The four chains of Torpedo receptor were immunologically distinct from one another and from higher molecular weight chains found in electric organ membranes. Antibodies to these chains reacted very efficiently with native Torpedo receptor, but the reverse was not true. Antibodies to native receptor from Torpedo and Electrophorus reacted slightly with each of the chains of the corresponding receptor. However, cross-reaction between chains and antibodies to any native receptor was most obviuos with the alpha chain of Torpedo or the corresponding alpha' chain of Electrophorus. Antiserum to alpha chains exhibited higher titer aginst receptor from denervated rat muscle. Antibodies from myasthenia gravis patients did not cross-react detectably with 125I-labeled chains from electric organ receptors. Most interspecies cross-reaction occurred at conformationally dependent determinants whose subunit localization could not be determined by reaction with the denatured chains.

Animals↗
PubMed1978

Immunization of rats with polypeptide chains from torpedo acetylcholine receptor causes an autoimmune response to receptors in rat muscle.

Four polypeptide chains were purified from acetylcholine receptor of Torpedo californica electric organ. Their apparent molecular weights were 64,000, 57,000, 49,500, and 38,000. Rats immunized with any of the four chains produced antibodies that crossreacted with rat muscle receptors in vivo. Specificities of anti-chain sera were evaluated in vitro by reaction with native receptor solubilized from electric organs and muscles of several species and by inhibition of this reaction with the purified polypeptide chains. The chains are immunologically distinct from one another. Antigenic determinants comparable to each chain of torpedo receptor are found in receptor from both rat and human muscle. At least part of each of these determinants is exposed on the extracellular surface of the muscle membrane. The most immunogenic determinants on native receptor are lost on denaturation to polypeptide chains. Its component peptides are much less immunogenic than native receptor, and induce antibodies of different specificity. Anti-receptor antibodies of many specificities can cause experimental autoimmune myasthenia gravis.

Acetylcholine↗
PubMed1978

Association of HLA-B8, DRw3, and anti-acetylcholine receptor antibodies in myasthenia gravis.

Twenty-eight patients with myasthenia gravis (MG), five with and 23 without thymoma, and 47 normal controls were typed for serologically defined HLA-A, B, C, and DRw antigens. Sera from all patients were titered for antibodies to acetylcholine receptors (AChR). The frequency of HLA-B8 and DRw3 in the non-thymoma MG patients was significantly higher than in the normal population. Most of the non-thymoma patients with AChR titers higher than the average level were positive for HLA-B8 and/or DRw3, while the majority of the HLA-B8(-) and/or DRw3(-) non-thymoma patients demonstrated AChR titers below average. These findings support the possibility of the existence of immune response genes in the HLA-B, DRw segment of the major histocompatibility complex which are concerned in the response to or recognition of autoantigens.

Acetylcholine↗