Solubilization of 20S acetylcholinesterase from chick retina

A 20S form of acetylcholinesterase has been solubilized from young chick retinas by means of a buffered salt-detergent solution containing EDTA. The release of this fast-sedimenting form of the enzyme is selectively blocked by the presence of even small amounts of Ca⁺⁺ in the homogenization medium. The collagen-tailed nature of this molecular species of acetylcholinesterase has been ascertained by collagenase digestion. This finding suggests that the avian central nervous system contains asymmetric, collagen-tailed quaternary structural forms of acetylcholinesterase as is the case in skeletal muscle and cholinergic ganglia.     

Interaction of Asymmetric and Globular Acetylcholinesterase Species with Glycosaminoglycans

Chicken muscle and retina, and rat muscle asymmetric acetylcholinesterase (AChE) species were bound to immobilized heparin at 0.4 M NaCl. Binding efficiency was between 50 and 80% for crude fraction I A-forms (AI; muscle), and nearly 100% for fraction II A-forms (AII; muscle and retina). Antibody-affinity-purified AI-forms (chicken) were, however, quantitatively bound to heparin-agarose gels, whereas diisopropylfluorophosphate-inactivated high-salt extracts partially prevented the binding of both AI and AII AChE forms, thus suggesting the presence in crude AI extracts of heparin-like molecules interfering with the tail-heparin interaction. All bound A-forms were progressively displaced from the heparin-agarose columns by increasing salt concentrations, with maximal release at about 0.6 M. They were also efficiently eluted by heparin solutions (1 mg/ml), other glycosaminoglycans being much less effective. Chicken globular AChE forms (G-forms, both low-salt-soluble and detergent-soluble) also bound to immobilized heparin in the absence of salt. Stepwise elution with increasing NaCl concentrations showed maximal release of G-forms at 0.15 M, all globular forms being totally displaced from the column at 0.4 M NaCl. Heparin (1 mg/ml) had the same eluting capacity as 0.4 M NaCl, whereas other glycosaminoglycans were only marginally effective. We conclude that the molecular forms of AChE in these vertebrate species interact with heparin, at salt concentrations that are characteristic for asymmetric and globular forms. Within the A and G molecular form groups, no differences were found in the behavior of the different fractions or subtypes, provided that the enzyme samples were free of interfering molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparin and the solubilization of asymmetric acetylcholinesterase

Heparin solubilizes asymmetric acetylcholinesterase, from chick skeletal muscle and retina, as a 24 S complex which is quantitatively converted to conventional asymmetric molecular forms of the enzyme (A12 and A8, either class I or class II) upon exposure to high salt. The simultaneous presence of salt and heparin in the homogenization medium selectively prevents, however, the release of class II A-forms in both muscle and retina. Heparin may generally act by displacing native proteoglycans involved in the attachment of the enzyme tail to the extracellular matrix, or its neural equivalent, being in turn removed by salt to yield typical asymmetric enzyme forms. Heparin would also appear to displace some other molecules specifically involved in the EDTA-sensitive attachment of class II tailed forms, this effect being antagonized by salt.     

Neural influence on the expression of acetylcholinesterase molecular forms in fast and slow rabbit skeletal muscles

With the aim of investigating the roles of motor innervation and activity on muscle characteristics, we studied the molecular forms of acetylcholinesterase (AChE) in fast-twitch (semimembranosus accessorius; SMa) and slow-twitch (semimembranosus proprius; SMp) muscles of the rabbit. We have shown that SMa and SMp express different patterns and tissue distribution of AChE forms and that the effect of long denervation varies with age. Three principal findings concerning expression of AChE molecular forms emerge from these studies. (1) The activity of AChE and the pattern of its molecular forms are particularly altered in adult denervated SMa and SMp muscles. AChE activity increases by 10-fold in both muscles, but asymmetric forms disappear in SMa and increase by 20-fold in SMp muscles. A similar alteration of AChE is found after tenotomy of these muscles, showing that the effect of denervation may be partly due to suppression of muscle activity. (2) The different changes occurring in the composition of AChE molecular forms in adult denervated SMa and SMp muscles are consistent with fluorescent staining with anti-AChE monoclonal antibodies and with DBA or VVA lectins, which bind to AChE asymmetric, collagen-tailed forms. These lectins poorly stain denervated SMa muscle surfaces but intensely stain neuromuscular junctions and extrasynaptic areas in denervated SMp muscle. (3) In contrast with the adult, denervation of 1-day-old muscles does not markedly modify the total amount of AChE or the proportions of its molecular forms, despite dramatic effects on muscle structure. These results are supported by studies of labeling with fluorescent DBA: the lectin only slightly stains the muscle fiber surface of denervated 15-day-old SMp muscle. Taken together, these data show that denervated muscles escape physiological regulation, producing increased levels of AChE with highly variable cellular distribution and patterns of molecular forms, depending on the age of operation and on the type of muscle.     

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagen-tailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagen-tailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQ-AChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process.     

Acetylcholinesterase molecular forms in the fast or slow muscles of the chicken and the pigeon

Molecular forms of acetylcholinesterase (AChE) were examined in various skeletal muscles of the chicken and the pigeon. In chicken pectoralis m., AChE was found to be restricted to endplate containing segments, and no asymmetric form could be detected in aneural samples. In the chicken muscles studied, a relation has been established between globular (G1,G2,G4) forms or asymmetric (A8,A12) forms, and muscle fibre types. Asymmetric forms are preponderant in fast-twitch muscles, whereas in slow tonic muscles 80% of the AChE activity is due to globular forms. However, comparison with pigeon muscles shows that AChE chicken muscle patterns may not be generalized.     

Isolation of the heparan sulfate proteoglycans from the extracellular matrix of rat skeletal muscle

We have previously shown that asymmetric collagen-tailed acetylcholinesterase (AChE) is anchored to the extracellular matrix (ECM) by heparan sulfate proteoglycans (HSPGs). Here we present our studies on the characterization of such PGs from the ECM of rat skeletal muscles. After radiolabeling with 35SO4 for 24h, PGs were extracted from the muscle ECM with 4.0 M guanidine-HCl containing protease inhibitors. PGs were subsequently isolated using sequential DEAE-Sephacel chromatography, digestion with chondroitinase ABC, and Sepharose CL-4B. Two different hydrodynamic size species of HSPGs were found. One type had a Mr of 4-6 X 10(5) (Kav = 0.25) as estimated by gel chromatography in the presence of 1% SDS and accounted for 75% of the total HSPGs. The other HSPG had a Mr 1.5-2.5 X 10(5) (Kav = 0.41). The glycosaminoglycan (GAG) side chains (Mr 20,000 and 12,000) were found composed only of heparan sulfate as determined by nitrous acid oxidation and heparitinase treatment. The large-sized HSPG, which is concentrated in synaptic regions, contains only GAG chains of Mr 20,000, suggesting that each HSPG contains only one kind of heparan sulfate chain in its structure. Our results definitively establish by biochemical criteria that the basement membrane of mammalian skeletal muscle contains HSPGs, the likely matrix receptor for the immobilization of the asymmetric collagen-tailed AChE at the neuromuscular junction.     

Amphiphilic and Nonamphiphilic Forms of Torpedo Cholinesterases: I. Solubility and Aggregation Properties

We report an analysis of the solubility and hydrophobic properties of the globular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) from various Torpedo tissues. We distinguish globular nonamphiphilic forms (Gna) from globular amphiphilic forms (Ga). The Ga forms bind micelles of detergent, as indicated by the following properties. They are converted by mild proteolysis into nonamphiphilic derivatives. Their Stokes radius in the presence of Triton X-100 is approximately 2 nm greater than that of their lytic derivatives. The G2a forms fall in two classes. Class I contains molecules that aggregate in the absence of detergent, when mixed with an AChE-depleted Triton X-100 extract from electric organ. AChE G2a forms from electric organs, nerves, skeletal muscle, and erythrocyte membranes correspond to this type, which is also detectable in detergent-soluble (DS) extracts of electric lobes and spinal cord. Class II forms never aggregate but only present a slight shift in sedimentation coefficient, in the presence or absence of detergent. This class contains the AChE G2a forms of plasma and of the low-salt-soluble (LSS) fractions from spinal cord and electric lobes. The heart possesses a BuChE G2a form of class II in LSS extracts, as well as a similar G1a form. G4a forms of AChE, which are solubilized only in the presence of detergent and aggregate in the absence of detergent, represent a large proportion of cholinesterase in DS extracts of nerves and spinal cord, together with a smaller component of G4a BuChE. These forms may be converted to nonamphiphilic derivatives by Pronase. Nonaggregating G4a forms exist at low levels in the plasma (BuChE) and in LSS extracts of nerves (BuChE) and spinal cord (AChE).     

Regulation of acetylcholinesterase synthesis and assembly by muscle activity. Effects of tetrodotoxin

The abundance and distribution of acetylcholinesterase (AChE) oligomeric forms expressed in skeletal muscle is strongly dependent upon the activity state of the cells. In this study, we examined several stages of AChE biogenesis to determine which ones were regulated by muscle activity. Inhibiting spontaneous contraction of tissue-cultured quail myotubes with tetrodotoxin (TTX) reduces AChE activity by approximately 30% of the levels found in actively contracting cells. This decrease is due primarily to the loss of 20 S asymmetric (collagen-tailed) AChE from TTX-treated cultures and is reflected in reduced pool sizes for both cell surface and intracellular AChE molecules. Using monoclonal anti-AChE antibodies to immunoprecipitate and quantify isotopically labeled enzyme molecules, we show that AChE down-regulation by TTX is not mediated through changes in the rates of synthesis or degradation of AChE polypeptide chains. Newly synthesized AChE polypeptides acquire enzymatic activity at the same rate in TTX-treated cultures as in actively contracting cells, however, a larger percentage of catalytically active dimers and tetramers are secreted from TTX-treated cultures compared with controls. These results suggest that TTX-induced down-regulation of asymmetric AChE occurs at the level of assembly of globular AChE molecules with collagen-like tail subunits in the Golgi apparatus, rather than through changes in the availability of catalytic subunits. Thus, post-translational mechanisms appear to play an important role in regulating the abundance and distribution of this important synaptic component in skeletal muscle.     

Analysis of a calcium ion binding system composed of two different sites

Using murexide (Mx), a metallochromic indicator, and a dual wavelength spectrophotometer with a high signal-to-noise ratio, the Ca⁺⁺ binding in a system containing two classes of binding sites was studied. Solutions with solute containing one or two classes of Ca⁺⁺ binding sites and without such solute were titrated with Ca⁺⁺ using Mx as an indicator of free Ca⁺⁺ concentration. Since curvilinear Scatchard plots are obtained from titration curves of solutes containing two classes of binding sites, a computer program was developed to resolve such plots into two linear partial plots, each corresponding to a single class of binding site. The validity of the procedure was examined with solutions of ethylene glycol bis(β-aminoethyl)-N-N′-tetraacetic acid, adenosine triphosphate (EGTA, ATP), or a mixture thereof. The method was also applied to biological material and it was found that a protein fraction isolated from rat skeletal muscle sarcotubular membranes, termed Fraction-2 (Fr-2), has two classes of binding sites for Ca⁺⁺; the association constants of the high affinity site and low affinity site are 4.3 × 10⁵ M^-1 and 9 × 10³ M^-1, respectively. The advantages and limitations of this methodology are discussed.     

Solubilization of collagen-tailed molecular forms of acetylcholinesterase from several brain areas in different vertebrate species

The relative efficiency of a buffered medium containing a high salt concentration and EDTA as a means to solubilize collagen-tailed molecular forms of acetylcholinesterase has been examined in four brain areas of several species belonging to different vertebrate classes. This extraction procedure has proved successful in most cases, with the yield of tailed enzyme varying between less than 1 and 26% of the total tissue activity. The solubilization values are consistently higher in more primitive vertebrates than in mammals and, for a given species, are usually lower in the telencephalon than in other brain structures. Our results confirm that the vertebrate central nervous system contains collagen-tailed quaternary structural forms of acetylcholinesterase.     

Interaction of calcium and local anesthetics with skeletal muscle microsomes

The influence of Ca⁺⁺, several drugs, and pH on the binding of Ca⁺⁺ by skeletal muscle microsomes was studied in vitro. A mass-law graphic analysis revealed the presence of three distinct species of Ca⁺⁺-binding sites in the microsomes, and the binding at only one of these sites was antagonized by local anesthetics and quinidine. These drugs also decreased the maximum Ca⁺⁺-binding capacity of the microsomes. Caffeine and ouabain exerted no effect on the binding at any of the sites. Procaine was also bound by microsomes, and this binding was antagonized by Ca⁺⁺, which also decreased the maximum procaine-binding capacity of microsomes. The sites that bind procaine and Ca⁺⁺ are not identical because the maximum-binding capacities of the interacting sites are distinctly different. The influence of pH on the ability of drugs to antagonize Ca⁺⁺ binding indicates that the displacing activity increases as the percentage of the drug in the nonionized form increases. All of the data obtained in the above studies are consistent with the interpretation that quinidine and local anesthetics of the procaine type noncompetitively antagonize the binding of Ca⁺⁺ by microsomes. The pharmacological significance of a noncompetitive interaction may be related to the property of local anesthetics and quinidine to increase contractile tension in skeletal muscle rather than to their ability to stabilize the cell membrane.     

Characterization of homologous divalent metal ion binding sites of vertebrate and molluscan myosins using electron paramagnetic resonance spectroscopy.

The binding of Ca²⁺, Mg²⁺ and Mn²⁺ to myosins from rabbit skeletal muscle, scallop striated adductor muscle and clam adductor muscle has been investigated. All three myosins bind two moles of divalent metal ion non-specifically and with high affinity (Mn²⁺ > Ca²⁺ > Mg²⁺). In addition, the molluscan myosins bind about a further two moles of Ca²⁺ specifically. Although rabbit myosin binds some Ca²⁺ in the presence of an excess of free Mg²⁺, this binding occurs at the nonspecific sites and should not be taken as evidence for a myosin-linked regulatory system of the type found in molluscan muscles. If such a system exists in vertebrate skeletal muscle, the homologous Ca²⁺-specific sites must be lost during the early stages of the myosin preparation.The characteristic electron paramagnetic resonance spectrum of the bound Mn²⁺ was utilized to confirm the homology of the non-specific sites in vertebrate and molluscan myosins. The sites are located on the “regulatory” class of light chain. Mn²⁺ bound to scallop myosin has a broad electron paramagnetic resonance spectrum, in contrast to the well-resolved spectra that it gives when bound to many other myosin species. This situation was exploited to identify homologous nonspecific, divalent metal-ion sites on the regulatory light chains from a variety of muscle types, including frog skeletal, rabbit cardiac, chicken gizzard and molluscan adductor muscles. When these light chains are combined with desensitized scallop myofibrils the electron paramagnetic resonance spectra of Mn²⁺ bound to the resultant hybrids are dominated by the signal from the non-specific site of the foreign regulatory light chain.     

Co-solubilization of asymmetric acetylcholinesterase and dermatan sulfate proteoglycan from the extracellular matrix of rat skeletal muscles

We have previously communicated that heparin released asymmetric acetylcholinesterase (AChE) from cholinergic synapses. Here we report studies showing that heparin, besides releasing asymmetric AChE from the skeletal muscle extracellular matrix (ECM), specifically solubilizes a dermatan sulfate proteoglycan (DSPG) which accounts for more than 95% of the 35S-released material. The co-solubilization of AChE and the proteoglycan opens up the possibility that both macromolecules could be involved in the formation of the soluble AChE complex observed after incubation of muscle homogenate with heparin. Our results suggest a possible association between asymmetric AChE and DSPG at the muscle ECM, moreover this work is the first report of the existence of DSPG at the skeletal muscle cell surface.     

Co-localization of nitric oxide synthase I (NOS I) and NMDA receptor subunit 1 (NMDAR-1) at the neuromuscular junction in rat and mouse skeletal muscle

Nitric oxide synthase I (NOS I) has been localized to the skeletal muscle sarcolemma in a variety of vertebrate species including man. It is particularly enriched at neuromuscular junctions. Recently, the N-methyl-d-aspartate (NMDA) receptor subunit 1 (NMDAR-1) has been detected in the postjunctional sarcolemma of rat diaphragm, providing a clue as to the possible source of Ca²⁺ ions that are necessary for NOS I activation. To address this possibility, we studied the distribution of NMDAR-1 and NOS I in mouse and rat skeletal muscles by immunohistochemistry and enzyme histochemistry. NMDAR-1 and NOS I were closely associated at neuromuscular junctions primarily of type II muscle fibers. NOS I was also present in the extrajunctional sarcolemma of this fiber type. Dystrophin, β-dystroglycan, α-sarcoglycan, and spectrin were found normally expressed in both the junctional and extrajunctional sarcolemma of both fiber types. By contrast, in the muscle sarcolemma of MDX mice, dystrophin and dystrophin-associated proteins were reduced or absent. NOS I immunoreactivity was lost from the extrajunctional sarcolemma and barely detectable in the junctional sarcolemma. NOS I activity was clearly demonstrable in the junctional sarcolemma by NADPH diaphorase histochemistry, especially when the two-step method was used. NMDAR-1 was not altered. These data suggest that different mechanisms act to attach NOS I to the junctional versus extrajunctional sarcolemma. It may further be postulated that NMDA receptors are involved not only in the regulation but also sarcolemmal targeting of NOS I at neuromuscular junctions of type II fibers. The evidence that glutamate may function as a messenger molecule at vertebrate neuromuscular junction is discussed.     

Studies on the in Vitro Interaction of Electrical Stimulation and Ca++ Movement in Sarcoplasmic Reticulum

Sarcoplasmic reticulum fragments (S.R.F.) were isolated from skeletal and heart muscles. These fragments were found to take up Ca⁺⁺ very actively from media. When monophasic square waves were passed through the S.R.F. suspension, the Ca⁺⁺ uptake by S.R.F. was decreased. When the suspension was stimulated electrically after the Ca⁺⁺ was taken up by S.R.F., the initiation and the cessation of the stimulation were followed by the release and re-uptake of Ca⁺⁺ by S.R.F., respectively. The degree of inhibition of the Ca⁺⁺ uptake as well as of the Ca⁺⁺ release by electrical stimulation was dependent on the voltage and the frequency of stimulation. The presence of inorganic phosphate or oxalate modified the influence of electrical stimulation on the release and the uptake of Ca⁺⁺ by S.R.F. Attempts were made to observe the release of Ca⁺⁺ by electrical stimulation from unfractionated sarcoplasmic reticulum remaining in myofibers, and the interaction of the released Ca⁺⁺ with myofibrils in vitro. For this purpose, the glycerol-extracted fiber was selected as a muscle model, since it contains both sarcoplasmic reticulum and myofibrils. It was found that electrical stimulation of skeletal and heart glycerol-extracted fibers resulted in the contraction of fibers. It appeared that the contraction of glycerol fibers by electrical stimulation was caused by the Ca⁺⁺ release from sarcoplasmic reticulum by stimulation.     

Calcium and facilitation at two classes of crustacean neuromuscular synapses

The closer muscle of the crab, Chionoecetes, has at least two classes of excitatory neuromuscular synapses. In one class of synapses an action potential depolarizing the synaptic region releases much more transmitter if it has been preceded recently by another action potential. The other class of synapses shows this property, called facilitation, to a far lesser extent. Immediately after one conditioning stimulus the level of facilitation is similar in both classes. The rate of the ensuing decay of the facilitation is the critical factor differentiating the two classes of synapses. The relationship between external Ca⁺⁺ concentration and transmitter release is similar for both classes of synapses. The slope of a double logarithmic plot of this relationship varies from 3.1 between 5 and 10 mM Ca⁺⁺ to 0.9 between 30 and 40 mM Ca⁺⁺. Facilitation does not significantly change when tested in external Ca⁺⁺ concentrations ranging from 7 to 30 mM. The extracellularly recorded nerve terminal action potential does not increase in amplitude during facilitation. The results suggest that the mechanism of synaptic facilitation is similar for both classes of synapses and occurs after the stage in transmitter release involving Ca⁺⁺.     

Observation of calcium uptake by isolated sarcoplasmic reticulum employing a fluorescent chelate probe

The fluorescent chelate probe technique is employed to observe the accumulation and binding of Ca⁺⁺ to isolated sarcoplasmic reticulum from skeletal and cardiac muscle. Chlorotetracycline serves as a fluorescent chelate probe which chelates to membrane bound Ca⁺⁺ giving rise to an intensely fluorescence adduct. An increase in fluorescence of chlorotetracycline is caused by ATP induced Ca⁺⁺ transport in both skeletal and cardiac muscle microsomes. The fluorescence spectra indicate that Ca⁺⁺ lies on the membrane surface in a relatively polar environment.     

Synaptic acetylcholinesterase of chicken muscle changes during development from a hybrid to a homogeneous enzyme.

The asymmetric (20S) form of acetylcholinesterase (AChE) in 1-day-old chick muscle is a hybrid enzyme containing both AChE (110 kd) and butyrylcholinesterase (BuChE, 72 kd) catalytic subunits. However, we now report that the asymmetric AChE extracted or immunopurified from older adult chicken muscles, where it is the endplate form, shows a progressive developmental loss of the BuChE subunit and its activities, centred around 4 weeks of age, while the AChE and collagenous subunits remain. In confirmation, using differential labelling and co-sedimentation it was shown that the hybrid 20S AChE/BuChE form of 1-day chick muscle is gradually and completely replaced during muscle maturation by a 21.3S form, also collagen-tailed but otherwise homogeneous in AChE catalytic subunits. Two other changes occur concomitantly. Firstly, the AChE catalytic subunit of the adult form has a lower apparent mol. wt in gel electrophoresis, by 5 kd, than the same subunit in the 1-day hybrid enzyme; this difference does not reside in the carbohydrate attachments. Secondly, the collagen tail changes, in that some conformation-dependent epitopes on it disappear in the same period. Hence, a major reorganization of the asymmetric AChE, involving all three types of subunit, occurs in the course of muscle development.     

Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: Developmental profiles,in Vivo and in cell culture,and recovery after inactivation

1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.