Human red blood cells are lysed by the neutrophil-derived oxidant hypochlorous acid (HOCl), although the mechanism of lysis is unknown. Hypobromous acid (HOBr), a similarly reactive oxidant, lysed red cells approx. 10-fold faster than HOCl. Therefore we compared the effects of these oxidants on thiols, membrane lipids and proteins to determine which reactions are associated with lysis. There was no difference in the loss of reduced glutathione or membrane thiols with either oxidant, but HOBr reacted more readily with membrane lipids and proteins. Bromohydrin derivatives of phospholipids and cholesterol were seen at approx. one-tenth the level of oxidant than chlorohydrins were. However, these products were detected only with high concentrations of HOCl or HOBr, which caused instant haemolysis. Membrane protein modification occurred at much lower doses of oxidant and was more closely correlated with lysis. SDS/PAGE analysis showed that band 3, the anion transport protein, was lost at the lowest dose of HOBr and at the higher concentrations of HOCl. Labelling the red cells with eosin 5-maleimide, a fluorescent label for band 3, suggested possible clustering of this protein in oxidant-exposed cells. There was also irreversible cross-linking of all the major membrane proteins; this reaction occurred more readily with HOBr. The results indicate that membrane protein modification is the reaction responsible for HOCl-mediated lysis. These effects, and particularly cross-link formation, might result in clustering of band 3 and other membrane and cytoskeletal proteins to form haemolytic pores.

Abstract. To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43-and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87-and 270-kD proteins were cleaved by Ca++-activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.

The primary structure of the 43-kilodalton peripheral membrane protein (43-kDa protein) of Torpedo nicotinic postsynaptic membrane has been determined. The 43-kDa protein, which was isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has an amino terminus resistant to Edman degradation, while the sequence at the carboxyl terminus is Tyr-Val. An amino acid sequence of 405 residues was obtained by NH2-terminal sequence analysis of complementary peptides generated by digestion with trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and endoproteinase Lys-C, as well as by chemical cleavage at methionine. This sequence of molecular mass 45,618 daltons lacks the amino terminus but extends to the carboxyl terminus of the 43-kDa protein. Unusual structural features of the 43-kDa protein include two regions of approximately 80 residues, each containing 10% cysteine, as well as stretches predicted to exist as amphipathic alpha-helices. Other than the group blocking the amino terminus, no evidence was found for posttranslational modification of amino acids. The 43-kDa protein may represent a novel protein family because a computer search of this sequence with the National Biomedical Research Foundation data base (Release 12.0) did not reveal any significant homology to known protein sequences.

Abstract. The synapse-specific Mr 43,000 protein (43K protein) and the acetylcholine receptor were visualized by freeze-etch immunoelectron microscopy in preparations of purified Torpedo postsynaptic membranes. Vesicles were immobilized on glass and then sheared open by sonication to expose the cytoplasmic surface. Membranes were labeled with monoclonal antibodies to the 43K protein or the acetylcholine receptor. The cytoplasmic surface was devoid of filamentous structure, and the 43K protein and the cytoplasmic projection of the acetylcholine receptor were associated with prominent surface particles. Acetylcholine receptor and 43K protein, in membrane surfaces in direct contact with glass coated with polyornithine, segregated into dense particle aggregates separated by smooth membrane patches, whereas those in contact with glass coated with Alcian Blue underwent little or no detectable rearrangement. After treatment of vesicles at alkaline pH to remove the 43K protein, the cytoplasmic surfaces were still covered by a dense array of particles that were more uniform in shape and appeared slightly shorter than those seen on unextracted membranes, but similar in height to the extracellular projection. Monoclonal antibodies to the acetylcholine receptor labeled these particles, while antibodies to 43K protein did not. We conclude that the 43K protein is in direct association with the receptor and that complexes of the receptor and 43K protein can undergo surface-induced lateral redistribution. In addition, the cytoplasmic projection of the acetylcholine receptor is sufficiently large to be readily detected by freeze-etch electron microscopy and is similar in height to the extracellular projection.

Postsynaptic membranes isolated from Torpedo electric organ are highly enriched in the nicotinic acetylcholine receptor and a nonreceptor protein of 43 kDa; the distribution of the 43-kDa protein and the receptor is coextensive in the electrical membrane. As During the formation of the neuromuscular junction, the postsynaptic membrane, just 0.1% of the total muscle fiber surface, undergoes morphological and biochemical specialization, distinguishing it from the extrasynaptic membrane. This specialization involves the preferential localization of acetylcholine receptor (AcChoR) and acetylcholinesterase, as well as other less well-characterized proteins (1). Some of the synapse-specific proteins were first described in the Torpedo electric organ where the electrocytes (electrical cells that compose the electric organ) are richly innervated by cholinergic neurons. The presence of synapse-specific proteins in the postsynaptic membrane of both the neuromuscular junction and the Torpedo electric organ implies that these proteins are involved in the function and maintenance of nicotinic cholinergic synapses.The AcChoR and a nonreceptor protein of 43 kDa are by far the major proteins found in highly purified postsynaptic membranes isolated from Torpedo electric organ (2). The 43-kDa protein is a peripheral protein on the cytoplasmic surface (3-5). Within the electrocyte, the distribution of 43-kDa protein is coextensive with AcChoR (6), and these two proteins are present in equimolar amounts (7). The 43-kDa protein (also called v1) is biochemically and immunologically distinct from creatine kinase (v2) and actin (v3) (8), two proteins that can have mobilities similar to that of the 43-kDa protein on NaDodSO4/polyacrylamide gels. Despite much speculation, the function of the 43-kDa protein remains unknown. It is proposed that the 43-kDa protein is a cytoskeletal component that anchors the AcChoR at the subsynaptic membrane (9-12) because the rotational movement of the receptor is increased in the absence of the 43-kDa protein (12). Also, crosslinking reagents can cause the oligomerization of 43-kDa protein (10,13,14) and the crosslinking of 43-kDa protein to the p-subunit of the AcChoR (10). The 43-kDa protein has also been reported to be an actin-binding protein (11) and a protein kinase (15,16).Immunological studies have found that 43-kDa protein, or a protein with cross-reacting antigenic determinants, is also present and colocalizes with AcChoR at rat, Xenopus, and chick neuromuscular junctions (17-19; G. D. Fischbach and J.B.C., unpublished observations). In particular, anti-43-kDa-protein reactive immunofluorescence was specifically associated with newly formed AcChoR clusters on the surface of Xenopus muscle cells in culture (18,19). These observations are consistent with the idea that 43-kDa protein is associated with the AcChoR at vertebrate muscle synapses. We are interested in the regulation of the subset of muscle genes that code for synapse-specific proteins. Here we report the isolation and sequen...