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Z W Hall

Publications and source records attributed to Z W Hall.

At least 91 records · Page 5Linked to original sources
PubMed1981

Topographical segregation of old and new acetylcholine receptors at developing ectopic endplates in adult rat muscle.

We have used radioautographic methods to examine the topography of addition and removal of acetylcholine receptors (AChRs) within receptor clusters at developing ectopic synapses in adult rat soleus muscle. After AChRs within a cluster had been pulse-labeled with 125I-alpha-bungarotoxin (125I-alpha-BuTx), the area that they occupied within the cluster shrank with time. Thus the old receptors at new endplates occupy a continually decreasing area of the growing receptor cluster. To localize newly added AChRs, we pretreated the muscles with unlabeled alpha-BuTx, thus blocking the old receptors, and then labeled newly added receptors with 125I-alpha-BuTx 1 or 2 d later. In radioautographs, AChR clusters from these muscles appeared as annuli or "doughnuts," unlike control (unpretreated) clusters, which were more nearly uniformly labeled. This visual impression was confirmed by analyzing the radial grain density distribution. Thus growth and turnover of AChR clusters at ectopic endplates takes place by the addition of receptors at the periphery of the clusters. Our data are most consistent with a model in which receptor removal occurs by endocytosis randomly throughout the cluster.

Acetylcholine↗
PubMed1981

Cytoplasmic actin in postsynaptic structures at the neuromuscular junction.

We used an antibody prepared against Aplysia (mollusc) body-wall actin that specifically reacts with certain forms of cytoplasmic actin in mammalian cells to probe for the presence of actin at the neuromuscular junction. Immunocytochemical studies showed that actin or an actinlike molecule is concentrated at neuromuscular junctions of normal and denervated adult rat muscle fibers. Actin is present at the neuromuscular junctions of fibers of developing diaphragm muscles as early as embryonic day 18, well before postsynaptic folds are formed. These results suggest that cytoplasmic actin may play a role in the clustering or stabilization of acetylcholine receptors at the neuromuscular junction.

Actins↗
PubMed1981

Cellular localization of the molecular forms of acetylcholinesterase in rat pheochromocytoma PC12 cells treated with nerve growth factor.

In rat pheochromocytoma (PC12) cells treated with nerve growth factor (NGF), there are several molecular forms of the enzyme acetylcholinesterase (AChE) which sediment on sucrose density gradients at 4 to 6, 10, and 16 S, respectively. We have investigated the cellular localization of these forms in PC12 cells. In order to determine which forms are soluble and which are membrane bound, we extracted PC12 cells in buffers of various ionic strengths and detergent compositions. To distinguish internal from external forms of the enzyme, we examined the effect of di-isopropyl fluorophosphate and BW284c51 dibromide, membrane-permeable and -impermeable inhibitors of AChE, respectively, AChE forms in intact cells. We also determined the susceptibility of the forms in intact cells to collagenase treatment. Based on these studies, we conclude that the globular G1 and G2 (4 to 6 S) forms are internal and consist of both soluble and membrane-associated species. Thirty percent of the G4 (10 S) form is bound to cytoplasmic membrane structures, while the remainder occurs as an integral component of the plasma membrane. The asymmetric A12 (16 S) form is also a surface protein but is extracted by high salt without detergent and is released from intact cells by collagenase. This form thus contains a collagenous domain and is located outside of the plasma membrane, where it may be associated with an extracellular matrix.

Acetylcholinesterase↗
PubMed1980

In situ labeling of Torpedo and rat muscle acetylcholine receptor by a photoaffinity derivative of alpha-bungarotoxin.

We synthesized a photoaffinity derivative of 125I-alpha-bungarotoxin that binds tightly and specifically to the acetylcholine receptor. We used this reagent to label the receptor in membranes from Torpedo electric organ and rat muscle and have analyzed the labeled polypeptides by immunoprecipitation and sodium dodecyl sulfate-gel electrophoresis. Reaction of the modified toxin with the Torpedo receptor in situ covalently labels the same four polypeptides as seen in Torpedo receptor preparations after solubilization and purification. The toxin derivative labels five polypeptides in muscle membranes that correspond in molecular weight to the subunits of the purified muscle receptor. These results provide evidence that, for both Torpedo and muscle receptors, the polypeptides associated with the purified protein also form part of the receptor in situ.

Affinity Labels↗
PubMed1979

Subunit structure and peptide mapping of junctional and extrajunctional acetylcholine receptors from rat muscle.

We have purified the junctional acetylcholine receptor from normal rat skeletal muscle and compared its structure with that of the extrajunctional receptor from denervated muscle. The two receptors from leg muscle were distinguished by isoelectric focusing and by reaction with sera from patients with myasthenia gravis. The junctional form of the acetylcholine receptor was purified from normal leg muscle by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose followed by sucrose gradient centrifugation. Analysis of radioiodinated receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the subunit structure of the junctional receptor was similar to that previously determined for the extra-junctional form (Froehner, S. C., et al. (1977) J. Biol. Chem. 252, 8589-8596), with major polypeptides, whose apparent molecular weights in 9% polyacrylamide gels were 45 000 and 51 000. In addition, several minor polypeptides were found. When the two receptors were labeled with different isotopes of iodine and run together on a sodium dodecyl sulfate gel, the subunits of one receptor could not be resolved from those of the other. As seen earlier with the extrajunctional form, the affinity alkylating reagent [3H]MBTA labeled the 45 000- and 49 000-dalton polypeptides of the junctional receptor. Peptide mapping showed that the two MBTA binding subunits are structurally related, although they are unrelated to the other polypeptides, and that the 45 000- and 51 000-dalton polypeptides of the junctional receptor were indistinguishable from those of the extrajunctional receptor. In addition, peptide mapping of the four subunits of acetylcholine receptor isolated from Torpedo californica electric organ showed that these four polypeptides appear to be structurally unrelated.

Acetylcholine↗
PubMed1979

Antibodies from patients with myasthenia gravis recognize determinants unique to extrajunctional acetylcholine receptors.

We have examined the interaction between sera from patients with myasthenia gravis and acetylcholine receptor (AcChoR) purified from normal and denervated rat skeletal muscles [junctional receptor (JR) and extrajunctional receptor (EJR), respectively]. Eight of ten myasthenic sera had titers against EJR that were significantly higher (1.1-2.4 times) than their titers against JR. The antireceptor titers of these sera ranged from 2 to 102 nM. Although activities of three other sera were too low (less than 1 nM) to allow accurate titrations, provisional measurements with these sera gave titers against EJR that were at least as high (1.0-1.4 times) as those against JR. Competition experiments with myasthenic sera demonstrated two classes of determinants on rat AcChoR: those that are common to JR and EJR and those that are present or exposed only on EJR. Myasthenic sera did not recognize any determinants unique to JR. Several antisera raised to purified AcChoR from eel or Torpedo electric organs or denervated rat skeletal muscle had equal titers against the two forms of receptor. Treatment of JR and EJR by various enzymatic or chemical procedures designed to alter prosthetic groups on the proteins failed to affect their antigenic reactivity. AcChoR from embryonic rats was indistinguishable immunologically from EJR of adult muscle.

Animals↗
PubMed1979

Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina.

Basal lamina (BL) ensheathes each skeletal muscle fiber and passes through the synaptic cleft at the neuromuscular junction. Synaptic portions of the BL are known to play important roles in the formation, function, and maintenance of the neuromuscular junction. Here we demonstrate molecular differences between synaptic and extrasynaptic BL. We obtained antisera to immunogens that might be derived from or share determinants with muscle fiber BL, and used immunohistochemical techniques to study the binding of antibodies to rat skeletal muscle. Four antisera contained antibodies that distinguished synaptic from extrasynaptic portions of the muscle fiber's surface. They were anti-anterior lens capsule, anti-acetylcholinesterase, anti-lens capsule collagen, and anti-muscle basement membrane collagen; the last two sera were selective only after antibodies binding to extrasynaptic areas had been removed by adsorption with connective tissue from endplate-free regions of muscle. Synaptic antigens revealed by each of the four sera were present on the external cell surface and persisted after removal of nerve terminal. Schwann cell, and postsynaptic plasma membrane. Thus, the antigens are contained in or connected to BL of the synaptic cleft. Details of staining patterns, differential susceptibility of antigens to proteolysis, and adsorption experiments showed that the antibodies define at least three different determinants that are present in synaptic but not extrasynaptic BL.

Acetylcholinesterase↗
PubMed1977

Subunit structure of the acetylcholine receptor from denervated rat skeletal muscle.

Acetylcholine receptor from denervated rat leg muscle was purified 48,000-fold by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose. A control preparation containing only contaminants was made by performing a parallel purification in which alpha-bungarotoxin was added to the preparation prior to the cobrotoxin/Sepharose step. Comparison of the receptor was greater than 90% pure. Examination of the purified receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two major polypeptide chains with apparent weights of 45,000 and 51,000, along with minor components of 49,000, 56,000, 62,000, and 110,000. Polypeptides of the same molecular weight were found when purified acetylcholine receptor was radioiodinated and further purified by sucrose gradient sedimentation. Analysis of receptor purified from denervated muscles that had been incubated with [35S]methionine in organ culture showed that all of the identified polypeptide chains were radioactive, indicating that they had been synthesized de novo after denervation.

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↗
PubMed1977

Factors influencing degradation of extrajunctional acetylcholine receptors in skeletal muscle.

During development and after both denervation and reinnervation in adult mammalian skeletal muscle, the level of acetylcholine (ACh) receptors in the extrajunctional membrane undergoes wide variation. We have determined the rate of extrajunctional receptor degradation in denervated muscles in organ culture under a variety of conditions by measuring the rate at which alpha-bungarotoxin bound to the receptors is degraded. Direct electrical stimulation of muscles for several days dramatically reduced the levels of extrajunctional ACh sensitivity, and also reduced the rate of receptor degradation. Since the effect of activity on the rate of receptor degradation is in the opposite direction of the observed change in receptor levels, we conclude that activity must also decrease the rate of receptor synthesis. Receptor degradation was also examined in muscles at various times after denervation. The half-time of degradation increased from approximately 7 hr at 2-5 days after denervation to approximately 14 hr at 10-14 days. Hypophysectomy, which decreases the average rate of protein degradation in muscle, also decreased the rate of extrajunctional receptor degradation, but thyroxine, which restores the normal rate of overall protein breakdown in hypophysectomized animals, did not affect receptor breakdown. Since hypophysectomy did not increase the level of extrajunctional ACh receptors, it must also affect ACh receptor synthesis.

Animals↗
PubMed1975

Acetylcholine receptors in normal and denervated rat diaphragm muscle. I. Purification and interaction with [125I]-alpha-bungarotoxin.

Acetylcholine receptors have been purified from junctional regions of normal rat diaphragm muscle and from extrajunctional regions of denervated diaphragm. The reaction of purified receptors with [122I]-alpha-bungarotoxin has been investigated by kinetic methods. The toxin-receptor complexes dissociated in a biphasic manner at 35 degrees with a rapidly dissociating component (t1/2 = 4 hr) and a slowly dissociating component (t1/2 is greater than or equal to 100 hr). The association reaction between toxin and receptor did not obey simple second-order kinetics but could be analyzed in terms of two classes of binding sites corresponding to the two rates of dissociation. This treatment of the data allowed derivation of association rate constants for the two sites. Value obtained for the dissociation constants were 3.7 times 10(-10) and less than or equal to 0.4 times 10(-10) M for the junctional receptor and 1.7 times 10(-10) and is less than or equal to 0.2 times 10(-10) M for the extrajunctional receptor. In each case it is the more tightly binding component that associates and dissociates more slowly. Receptors present in crude preparations were comparable to purified receptors in their reaction with [125I-alpha-bungarotoxin. The validity of the two site model is discussed in relation to the kinetic studies.

Acetylcholine↗
PubMed1975

Acetylcholine receptors in normal and denervated rat diaphragm muscle. II. Comparison of junctional and extrajunctional receptors.

Acetylcholine (ACh) receptors have been purified separately from normal rat diaphragm muscle (junctional receptors) and from extrajunctional regions of denervated diaphragm (extrajunctional receptors) in order to compare their properties. The toxin-receptor complexes of the two receptors were indistinguishable by gel filtration and by zone sedimentation in sucrose gradients, and showed identical precipitation curves with rabbit antiserum to the eel ACh receptor. Both toxin-receptor complexes bind concanavalin A and are therefore probably glycoproteins. Low concentrations of d-tubocuratine (dTC) were more effective in decreasing the rate of toxin binding to junctional than to extrajunctional receptors. The apparent dissociation constant for dTC binding to the junctional receptor was 4.5 X 10 minus 8 M, whereas the value for the extrajunctional receptor was 5.5 X 10 minus 7 M. When the complexes were analyzed by isoelectric focusing, the junctional complex focused at approximately 0.15 pH unit lower than the extrajunctional complex. This result was also found with crude preparations of receptor. We conclude that junctional and extrajunctional receptors are similar but distinct molecules. The properties of receptors present in neonatal diaphragm muscle were also examined and found to be similar to those of receptors in denervated muscle, as shown by dTC inhibition and isoelectric focusing.

Acetylcholine↗