George Kemp

Juvenile myasthenia gravis

We studied 32 children with myasthenia gravis over a period of 12 years. The mean age at onset was 7.7 years. Presentation was ocular in 63% of patients. Another major disease in addition to myasthenia occurred in 44% of patients; a seizure disorder was the most commonly associated disease. Serum IgG antibody to nicotinic acetylcholine receptor was present in 53% of patients and did not correlate with severity of disease or treatment. Medical management was effective in 63%; thymectomy was effective in only 28%. We conclude that myasthenia gravis appears commonly before age 10 and is associated with the risk of some disease other than hyperthyroidism. Serum IgG nicotinic acetylcholine receptor antibody is present less frequently than in normal adults, and vigorous medical management should be attempted before thymectomy.

Purification and characterization of the α-bungarotoxin binding protein from rat brain

The α-bungarotoxin (BGT) binding protein from rat brain has been purified and its polypeptide chain composition has been examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polypeptide chains with Mrs of 55,000, 53,500 and 49,000 have been identified as constituents of the protein. The affinity ligand [3H]maleimidobenzyl trimethylammonium bromide ([3H]MBTA), used to identify the ligand binding site on neuromuscular junction acetylcholine receptors (NMJ AChRs), binds to the 55,000 dalton polypeptide chain. Using a technique where ligands are bound to the protein while the protein is immobilized on α-cobratoxin-Sepharose 4B, it was established that the brain BGT binding protein, like NMJ AChRs, possesses two binding sites for BGT. These experiments reinforce previous evidence that the brain BGT binding protein is closely related but not identical to NMJ AChRs.

Purification and characterization of nicotinic acetylcholine receptors from muscle

The nicotinic acetylcholine receptor was purified from normal and denervated rat skeletal muscle. The purification protocol included alpha-cobratoxin biospecific adsorption, ion exchange chromatography, and gel filtration steps. The highest specific activity achieved was 7.5 pmol of 125I-alpha-bungarotoxin binding sites per microgram protein. Sodium dodecyl sulfate gel electrophoresis of purified AChR revealed subunits with molecular weights of 42,000 and 66,000 daltons and a minor component with a molecular weight of 52,000 daltons. Normal muscle AChR is comprised of one toxin binding component. Upon denervation a second component appears, but both components are increased as a consequence of denervation. A dissociation constant of 1.5 x 10(-8)M was determined for d-tubocurarine from receptor from both normal and denervated muscle. A dissociation constant of 1 x 10(-7)M for acetylcholine, perhaps analogous to the high affinity acetylcholine binding observed in electric fish receptor, was determined.

INTERACTION OF HUMAN ANTIBODY AND MURINE MONOCLONAL ANTIBODY WITH MUSCLE ACETYLCHOLINE RECEPTOR

Immunological analyses of the nicotinic acetylcholine receptor (AChR) have led to important discoveries concerning its structure and function. The AChR is the target of an autoimmune response in myasthenia gravis (MG) , a debilitating neuromuscular disease. Antibodies which bind to the receptor can be detected in the serum of SO-90% of myasthenia However, the titer of anti-AChR immunoglobulins correlates poorly with disease severity 6 * suggesting a complex pathogenesis in MG. Myasthenic antibodies have been used to detect immunological differences between junctional receptor (JR) and extrajunctional receptor (EJR) isolated from mammalian EJR appears to contain antigenic determinants which are not found on JR. In addition, antireceptor antibodies have been employed to characterize the subunit composition of electric fish receptor. Antibodies against each of the four elasmobranch AChR polypeptide chains react with native AChR from Electrophorus electricus (electric eel) and mammalian skeletal muscle, suggesting that AChR from all three sources shares a common subunit structure.9 Animals immunized with purified AChR often display signs of weakness which are characteristic of human MG.10l1 These findings have led to the development of an animal model of the disease known as experimental myasthenia gravis (EMG) . Recently, monoclonal antibodies (MAbs) against the AChR have been produced by somatic cell hybridization techniques.12+ l3 MAbs have been shown to produce EMG in rats 14, l5 and guinea pigs.14 The studies described in this report were aimed at immunological characterization of the receptor and delineation of its role in MG. Specifically, we wanted to investigate the nature of the differences between JR and EJR and to examine the role of the different AChR subunits as antigenic sites in MG.

A novel acetylcholine receptor-related peptide blocks canine cardiac ganglia and inhibits the nicotinic receptor of PC-12 cells

A 13 amino acid peptide from the calf muscle acetylcholine receptor has been previously shown to bind both snake neurotoxins and acetylcholine. In the experiments reported here a modified complementary peptide (cAChR) derived from that acetylcholine receptor peptide was tested for biological activity in a canine heart preparation. It was expected that the modified complementary peptide would exhibit either acetylcholine-like effects or acetylcholine inhibiting effects, since, as a complementary peptide to the receptor, it should resemble acetylcholine. In these studies cAChR was administered via the sinus node artery of dog hearts in intact animals which were anesthetized with pentobarbital, intubated, and prepared with local cardiac electrograms and force gauges. cAChR was also injected directly into thoracic sympathetic ganglia. Both approaches demonstrated cAChR inhibition of neural transmission, cAChR was added to the medium of carbachol stimulated PC-12 cells. In these cells, derived from a rat pheochromocytoma, sodium flux is controlled by neural nicotinic receptors. With or without preincubation cAChR inhibited carbachol stimulation of sodium flux, exhibiting a Ki of approximately 9 x 10(-5) (similar to that of hexamethonium). Thus cAChR appears to be a novel synthetic peptide which interrupts nicotinic cholinergic neural transmission by acting as an antagonist of the neural nicotinic receptor.

Characterization of the α-Bungarotoxin Binding Protein from PC12 Cells

Nicotinic acetylcholine receptors (nAChRs) from electric fish electric organ and mammalian skeletal muscle have become the best characterized neurotransmitter receptors in the realm of neurobiology. They are composed of four kinds of polypeptide chains, designated α, β, γ, and, δ Mrs 50K, 54K, 56K, and 58K, respectively, for the unglycosylated chains, but 40K, 50K, 60K, and 65K by sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) (for review see Changeux, et al, 1984). These subunits appear to surround a central ligand-gated ion channel like barrel staves (Stroud and Finer-Moore, 1985). Each of the four subunits share extensive sequence homology, suggesting that they were duplicated from a single primordial gene (Raftery, et al, 1980, Noda, et al, 1983). The α-chain contains a high affinity binding site for elapid α-neurotoxins like α-bungarotoxin (BGT) and for other cholinergic ligands, and has an unusual conformationally strained and thus easily reducible disulfide bond between adjacent cysteine residues at positions 192–193 (Kao, et al, 1984). This disulfide bond is in close proximity to the ligand binding site and after reduction is labelled by the affinity-directed reagents bromoacetyl-[3H]choline [3H]BAC and 4-(N-maleimido)-[3H]benzyltrimethylammonium iodide ([3H]MBTA). Each receptor monomer contains two α-chains (and thus two high affinity ligand binding sites) and one each of the other three subunits.