Stanley H. Appel

Serum Globulin in Myasthenia Gravis: Inhibition of α-Bungarotoxin Binding to Acetylcholine Receptors

Serum factors that inhibit the binding of 125I-labeled α-bungarotoxin to the acetylcholine receptor extracted in detergent from denervated rat muscle were detected by a sensitive assay. The serum of at least 5 and possibly 11 out of 15 patients with myasthenia gravis showed inhibitory activity that was localized to the globulin fraction. No controls showed inhibitory activity. The demonstration of inhibitory globulins may help explain the involvement of the immune system in the pathophysiology of the neuromuscular junction in patients with myasthenia gravis.

Phosphorylation of component a of the human erythrocyte membrane in myotonic muscular dystrophy.

SummaryEndogenous membrane protein kinase activity in fresh erythrocyte ghosts is altered in myotonic muscular dystrophy. Phosphorylation of erythrocyte Component a, which migrates with an apparent molecular weight of 90,000 to 100,000, is significantly reduced compared to age- and sex-matched controls. The difference in endogenous membrane protein kinase activity in fresh RBC membranes lends confirmation to the suggestion that myotonic dystrophy is a disease of widespread membrane alterations.

Interaction of myasthenic serum globulin with the acetylcholine receptor.

A serum factor from patients with myasthenia gravis which inhibited the binding of 125I-labeled alpha-bungarotoxin to acetylcholine receptor extracted with Triton X-100 from rat muscle has been studied in detail. The inhibitory activity was localized to the IgG fraction based upon the fractionations by sodium sulfate precipitation and DEAE chromatography as well as reaction with anti-IgG globulin. The myasthenic globulin inhibited toxin binding to receptors extracted from degenerated muscle but did not inhibit toxin binding to normal junctional receptors. At saturation levels of myasthenic globulin, the number of denervated acetylcholine receptors available for toxin binding was reduced approx. 50 percent. The myastehnic globulin was found to bind to denervated acetylcholine receptors but not to normal acetylcholine receptors by a radioimmunoassay technique in which myasthenic globulin incubated with 125I-labeled alpha bungarotoxin-receptor complexes was precipitated by anti-IgG serum. The globulin binding was saturable over the same range as inhibition of toxin binding. The data suggest that the myasthenic IgC binds to a site on the receptor complex juxtaposed to the acetylcholine receptor site. The myasthenic globulin appears to be a useful probe for investigation differences between acetylcholine receptors extracted from normal and denervated muscle and for investigating the pathogenesis of myasthenia gravis.

Erythrocyte spectrin peak II phosphorylation in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a rapidly progressive crippling disease of young boys that is inherited as an X-linked recessive trait. Previous studies have demonstrated the usefulness of erythrocyte studies in exploring membrane abnormalities in inheritied muscular dystrophy. Erythrocyte spectrin peak II protein (m.w. equivalent to 220,000) was more highly phosphorylated under initial rate conditions in DMD than in controls. The extent of peak II phosphorylation was greater in DMD erythrocytes and a Na+ stimulated peak II phosphorylation effect (Avruch and Fairbanks 1974) was not found to account for the differences between DMD and controls. The phosphorylated state of spectrin proteins in the membrane was evaluated and no differences in DMD could be measured. The maximal transfer of phosphate from differences in DMD could be measured. The maximal transfer of phosphate from [gamma-32P]ATP to spectrin peak II accounts for approximately 5-10% of the total phosphate content of spectrin.

Increased phosphorylated components of erythrocyte membrane spectrin band II with reference to Duchenne muscular dystrophy

Increased erythrocyte band II phosphorylation has been demonstrated in patients with Duchenne muscular dystrophy (DMD). Band II has been purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing of purified band II from which SDS has been removed reveals heterogeneity. Band II migrates as multiple bands on isoelectric focusing gels and as a single band on SDS-polyacrylamide gels. The increased phosphorylation in DMD does not occur uniformly in all components of band II. Highly phosphorylated components of band II are present in DMD in which only minimal phosphorylation occurs in controls. These results suggest the presence of an abnormal band II substrate in erythrocyte membranes from DMD patients.

EFFECTS OF NORMAL AND MYASTHENIC SERUM FACTORS ON INNERVATED AND CHRONICALLY DENERVATED MAMMALIAN MUSCLES

Myasthenia gravis (MG) is a disease that is characterized by muscle weakness that becomes particularly apparent during repetitive motor unit activity. The mechanism underlying the progressive inability of the muscle to respond to repetitive neuronal discharge is still controversial. There is evidence that suggests that the amount of acetylcholine (ACh) released is insufficient and this insufficiency eventually results in the blockade of muscle contraction upon repetitive stimu1ation.l Using [1251]-a-bungarotoxin, Fambrough and associates have found that the density of cholinergic receptors in the postjunctional region was markedly reduced in muscles from myasthenic patients. This postsynaptic defect in receptor density was proposed to be of significant importance in explaining the decremental responses with repetitive stimulation. Recent studies suggest that an immunological defect related to the thymus may exist in myasthenic patients.3 The hypothesis of an immunological basis for myasthenia gravis was further supported by recent studies which demonstrated that sera and immunoglobulin G (IgG) fractions from diseased patients inhibited a-bungarotoxin (a-BuTx) binding to isolated extrajunctional receptor^.^ These findings prompted us to examine the effect of sera and immunoglobulin factors on membrane properties utilizing electrophysiological techniques. The results indicate that sera from myasthenic. patients do not directly influence the physiological activity of junctional or extrajunctional ACh receptors.

Phosphorylation of Muscle Membranes: Identification of a Membrane-Bound Protein Kinase

A membrane-bound protein kinase occurs in membranes derived from rat skeletal muscle and appears limited to a surface membrane fraction. The enzyme is magnesium dependent, is only minimally stimulated by cyclic nucleotides, and phosphorylates serine and to a lesser extent threonine residues of three membrane proteins with molecular weights of less than 30,000.

Cholinergic Sites in Skeletal Muscle

The physiological properties of mammalian skeletal muscle vary considerably as a function of innervation. Developmentally, the resting membrane potential, the cell surface distribution of acetyl-choline sensitivity and the characteristics of the action potential all change (1, 2, 25). Experimental denervation causes the muscles to revert to a state similar to the one observed prior to innervation ontogenetically. In recent years considerable effort has been directed towards the correlation of molecular properties of the membrane with denervation induced changes in physiological properties of the muscle (3, 5, 8–11, 15–17, 19, 21, 22). This approach, is pertinent to the understanding of both the molecular mechanisms of these physiological phenomena, and their significance to the innervation process.

Serum acetylcholine-receptor antibodies in myasthenia gravis.

The etiology as well as the pathogenesis of myasthenia gravis remain unknown. The clinical symptomatology has been attributed to a defect in transmission at the neuromuscular junction, which is physiologically manifest as a diminution in the amplitude of miniature end-plate potentials. Recent evidence supports a postsynaptic localization of the functional defect, although it is unclear to what extent alterations at the nerve terminal also contribute to the pathogenesis. The morphology of both sites is altered; and the gap between nerve terminal and muscle end plate is considerably widened. Drugs that function primarily at the nerve terminal (hemicholinium) simulate certain myasthenic features, but compounds that function at the postsynaptic membrane (e.g., cobra a-toxin) appear to simulate a larger number of such features. The normal depolarization of end-plate regions by decamethonium and carbachol applied in a bath as well as the normal response to the iontophoretic microapplication of acetylcholine have suggested a presynaptic origin of the On the other hand, the increased dose of intra-arterial acetylcholine required to alter the spontaneous end-plate potentials of opponens pollicis muscle of myasthenic patients has suggested that the primary defect is of postsynaptic origin.? A postsynaptic defect is also supported by a decrease in the binding of abungarotoxin to biopsies of myasthenic muscle.8 A complete explanation of the pathogenesis of myasthenia gravis must also encompass the immunological features of the condition. Thymic hyperplasia is present in a high percentage of patients,Q circulating antibodies which react with muscle proteins are present in some patients; lo lymphocytes toxic to muscle in vitro are noted in this disorder; l1 lymphatic drainage has been reported to be beneficial in the treatment of myasthenia gravis; l2 and a myasthenic-like syndrome has been produced experimentally in rabbits by the inoculation of electric eel acetylcholine receptor.13

Effects of denervation on glycoproteins of rabbit gastrocnemius and soleus muscles

Abstract Rabbit gastrocnemius and soleus differ in speed of contraction, in acetylcholine sensitivity, and in calcium uptake into fragmented sarcoplasmic reticulum. Following denervation, the gastrocnemius changes with respect to speed of contraction, acetylcholine sensitivity, and calcium uptake, whereas the soleus changes much less. Glycoproteins are important constituents of cell membranes, and it was felt that these constituents may reflect differences between innervated and denervated muscles. Concentration of protein-bound sialic acid was 0.75 nmoles/mg protein in fat free residue of innervated gastrocnemius and 1.4 nmoles/mg protein in fat free residue of innervated soleus. The concentration of protein-bound hexosamines was 4.0 nmoles/mg protein in gastrocnemius and 5.8 nmoles/mg protein in soleus. Following denervation for fifteen days, the protein-bound NANA ( N -acetylneuraminic acid) of gastrocnemius increased from 638 nmoles/muscle to 920 nmoles/muscle; whereas soleus protein-bound NANA did not change (168 nmoles/muscle versus 180 nmoles/muscle). The total protein bound hexosamines did not change significantly in either denervated gastrocnemius or soleus. The data suggest the synthesis de novo of sialic acid-containing glycoproteins or the enhanced transfer of sialic acid to preformed proteins in denervated gastrocnemius but not in soleus.