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Journal ArticleDOI

Globular and asymmetric acetylcholinesterase in frog muscle basal lamina sheaths.

01 Mar 1986-Journal of Cell Biology (Rockefeller University Press)-Vol. 102, Iss: 3, pp 762-768

TL;DR: Observations show that all acetylcholinesterase forms can be accumulated in frog muscle BL and in the motor endplate-rich region of control muscle.

AbstractAfter denervation in vivo, the frog cutaneus pectoris muscle can be led to degenerate by sectioning the muscle fibers on both sides of the region rich in motor endplate, leaving, 2 wk later, a muscle bridge containing the basal lamina (BL) sheaths of the muscle fibers (28). This preparation still contains various tissue remnants and some acetylcholine receptor-containing membranes. A further mild extraction by Triton X-100, a nonionic detergent, gives a pure BL sheath preparation, devoid of acetylcholine receptors. At the electron microscope level, this latter preparation is essentially composed of the muscle BL with no attached plasmic membrane and cellular component originating from Schwann cells or macrophages. Acetylcholinesterase is still present in high amounts in this BL sheath preparation. In both preparations, five major molecular forms (18, 14, 11, 6, and 3.5 S) can be identified that have either an asymmetric or a globular character. Their relative amount is found to be very similar in the BL and in the motor endplate-rich region of control muscle. Thus, observations show that all acetylcholinesterase forms can be accumulated in frog muscle BL.

Topics: Basal lamina (54%), Acetylcholine (51%), Denervation (51%)

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Citations
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Book ChapterDOI
01 Jan 1988
TL;DR: It is found useful first to classify them as molecular forms, according to their hydrodynamic parameters (sedimentation coefficient, Stokes radius), corresponding to different quaternary structures.
Abstract: Cholinesterases (ChEs) hydrolyse choline esters specifically and rapidly and thus play an essential role in cholinergic transmission, e. g. at the neuromuscular junctions of vertebrates. Vertebrates possess two distinct ChEs, acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase, also called pseudocholinesterase or non-specific cholinesterase (BuChE, EC 3.1.1.8). Both these enzymes present multiple molecular variants which possess identical catalytic activity but differ in their molecular structure and interactions. The molecular variants can be differentiated by various analytical procedures. We find it useful first to classify them as molecular forms, according to their hydrodynamic parameters (sedimentation coefficient, Stokes radius), corresponding to different quaternary structures.

90 citations


Journal ArticleDOI
TL;DR: N-CAM may play an important role not only in the determination of synaptic areas but also in Schwann cell-axon interactions during nerve regeneration, as suggested by results of an in vivo preparation of frog basal lamina sheaths obtained by inducing the degeneration of both nerve and muscle fibers.
Abstract: The neural cell adhesion molecule (N-CAM) is a membrane glycoprotein involved in neuron-neuron and neuron-muscle adhesion. It can be synthesized in various forms by both nerve and muscle and it becomes concentrated at the motor endplate. Biochemical analysis of a frog muscle extract enriched in basal lamina revealed the presence of a polydisperse, polysialylated form of N-CAM with an average Mr of approximately 160,000 as determined by SDS-PAGE, which was converted to a form of 125,000 Mr by treatment with neuraminidase. To define further the role of N-CAM in neuromuscular junction organization, we studied the distribution of N-CAM in an in vivo preparation of frog basal lamina sheaths obtained by inducing the degeneration of both nerve and muscle fibers. Immunoreactive material could be readily detected by anti-N-CAM antibodies in such basal lamina sheaths. Ultrastructural analysis using immunogold techniques revealed N-CAM in close association with the basal lamina sheaths, present in dense accumulation at places that presumably correspond to synaptic regions. N-CAM epitopes were also associated with collagen fibrils in the extracellular matrix. The ability of anti-N-CAM antibodies to perturb nerve regeneration and reinnervation of the remaining basal lamina sheaths was then examined. In control animals, myelinating Schwann cells wrapped around the regenerated axon and reinnervation occurred only at the old synaptic areas; new contacts between nerve and basal lamina had a terminal Schwann cell capping the nerve terminal. In the presence of anti-N-CAM antibodies, three major abnormalities were observed in the regeneration and reinnervation processes: (a) regenerated axons in nerve trunks that had grown back into the old Schwann cell basal lamina were rarely associated with myelinating Schwann cell processes, (b) ectopic synapses were often present, and (c) many of the axon terminals lacked a terminal Schwann cell capping the nerve-basal lamina contact area. These results suggest that N-CAM may play an important role not only in the determination of synaptic areas but also in Schwann cell-axon interactions during nerve regeneration.

83 citations


Cites background from "Globular and asymmetric acetylcholi..."

  • ...…pectoris muscle, the orientations of the basal lamina sheaths are preserved (Sanes et al., 1978) and the sites where synapses had been situated can be identified at the light (Letinsky et al., 1976) as well as at the electron microscopic level (Marshall et al., 1977; Nicolet et al., 1986)....

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  • ..., 1976) as well as at the electron microscopic level (Marshall et al., 1977; Nicolet et al., 1986)....

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  • ...with remnants of plasma membrane, which are very rarely found in this preparation (Nicolet et al., 1986)....

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  • ...The immunoreactive material was not associated with remnants of plasma membrane, which are very rarely found in this preparation (Nicolet et al., 1986 )....

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Book ChapterDOI
01 Jan 1988
TL;DR: It is shown that the two enzymes present globular forms which exist as non-hydrophobic as well as amphiphilic molecules, and asymmetric, collagen-tailed molecules, which may be further subdivided according to the binding of lectins or according to their electrophoretic migrations in nondenaturing electrophoresis.
Abstract: In Chapter 8 a we have described the molecular structure and the interactions of the multiple molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) in vertebrates. We show there that the two enzymes present globular forms (G1 G2, G4) which exist as non-hydrophobic as well as amphiphilic molecules, and asymmetric, collagen-tailed molecules (A4, A8, A12). These molecular forms may be further subdivided according to the binding of lectins or according to their electrophoretic migrations in nondenaturing electrophoresis.

57 citations


Book ChapterDOI
TL;DR: The two types of ChE (generic abbreviation for any Cholinesterase) are readily distinguished not only by their substrate specificity but also by their response to selective inhibitors.
Abstract: Acetylcholinesterase (acetylcholine acetylhydrolase, AChE: EC 3.1.1.7) and butyrylcholinesterase (acylcholine acylhydrolase, BuChE: EC 3.1.1.8) both possess the capacity to hydrolyze choline esters, although the latter accepts a much wider variety of substrates. These two enzymes are found in a large number of excitable and nonexcitable tissues in most species, including humans (for review see Silver, 1974). The two types of ChE (generic abbreviation for any Cholinesterase) are readily distinguished not only by their substrate specificity but also by their response to selective inhibitors.

43 citations


Journal ArticleDOI
TL;DR: The results suggest that cytotactin plays a primordial role in synaptogenesis, at least during nerve regeneration and reinnervation in the adult neuromuscular system.
Abstract: The expression of cytotactin, an extracellular matrix glycoprotein involved in morphogenesis and regeneration, was determined in the normal and regenerating neuromuscular system of the frog Rana temporaria. Cytotactin was expressed in adult brain and gut as two major components of Mr 190,000 and 200,000 and a minor form of higher molecular weight, but was almost undetectable in skeletal muscle extract. However, cytotactin was concentrated at the neuromuscular junctions as well as at the nodes of Ranvier. After nerve transection, cytotactin staining increased in the distal stump along the endoneurial tubes. In preparations of basal lamina sheaths of frog cutaneous pectoris muscle obtained by inducing the degeneration of both nerve and muscle fibers, cytotactin was found in dense accumulations at original synaptic sites. In order to define the role of cytotactin in axonal regeneration and muscle reinnervation, the effect of anti-cytotactin antibodies on the reinnervation of the basal lamina sheaths preparations was examined in vivo. In control preparations, regenerating nerve terminals preferentially reinnervate the original synaptic sites. In the presence of anti-cytotactin antibodies, axon regeneration occurred with normal fasciculation and branching but with altered preterminal nerve fibers pathways. Ultrastructural observations showed that synaptic basal laminae reinnervation was greatly delayed or inhibited. These results suggest that cytotactin plays a primordial role in synaptogenesis, at least during nerve regeneration and reinnervation in the adult neuromuscular system.

41 citations


References
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Journal ArticleDOI
TL;DR: A photometric method for determining acetylcholinesterase activity of tissue extracts, homogenates, cell suspensions, etc., has been described and Kinetic constants determined by this system for erythrocyte eholinesterases are presented.
Abstract: A photometric method for determining acetylcholinesterase activity of tissue extracts, homogenates, cell suspensions, etc., has been described. The enzyme activity is measured by following the increase of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion. It is based on coupling of these reactions: The latter reaction is rapid and the assay is sensitive (i.e. a 10 μ1 sample of blood is adequate). The use of a recorder has been most helpful, but is not essential. The method has been used to study the enzyme in human erythrocytes and homogenates of rat brain, kidney, lungs, liver and muscle tissue. Kinetic constants determined by this system for erythrocyte eholinesterase are presented. The data obtained with acetylthiocholine as substrate are similar to those with acetylcholine.

20,867 citations


Journal ArticleDOI
TL;DR: A histochemical method is presented for localizing ChE activity by incubating tissue sections in a medium containing acetylthiocholine, copper glycinate and copper thiocholine with results obtained with several tissues containing specific ChE.
Abstract: SummaryA histochemical method is presented for localizing ChE activity by incubating tissue sections in a medium containing acetylthiocholine, copper glycinate and copper thiocholine. Results obtained with several tissues containing specific ChE are described and illustrated.

1,171 citations


Journal ArticleDOI
TL;DR: The mechanism of cholinergic neurotransmission requires the rapid inac­ tivation of acetylcholine, which exists in all classes of vertebrates and is characterized in horse serum by Stedman et al (1932), who called it choli­ nesterase.
Abstract: The mechanism of cholinergic neurotransmission requires the rapid inac­ tivation of acetylcholine (Dale 1914). Loewi & Navratil showed in 1926 that acetylcholine can be destroyed by an enzyme that exists in aqueous extracts of frog tissues. An esterase that specifically hydrolyzes choline esters was characterized in horse serum by Stedman et al (1932), who called it choli­ nesterase. It was later found that blood cells also contain a high level of acetylcholine-hydrolyzing activity (Stedman & Stedman 1935). Alles & Hawes (1940) subsequently found that in human blood the serum and cell enzymes are qualitatively different; Mendel et al (1943a) showed that, al­ though the serum enzyme hydrolyzes butyrylcholine or propionylcholine faster than acetylcholine [as already noted by Stedman et al (1932)], the cell-bound enzyme acts preferentially on acetylcholine, at low substrate concentration. The particulate enzyme also presents a characteristic excess substrate inhibition, so that its activity varies, in a bell-shaped manner, as a function of substrate concentration (Mendel & Rudney 1943). These two activities exist in all classes of vertebrates. The serum enzyme (Be 3.1.1.8) varies somewhat in its specificity, notably in the relative rates of hydrolysis of propionylcholine and butyrylcholine (Augustinsson 1959a,b). The serum enzyme was called originally "nonspecific" cholineste­ rase, or "pseudocholinesterase" (Mendel et al 1943, Mendell & Rudney 1943). In contrast, the erythrocyte enzyme (EC 3.1.1.7) was considered the

797 citations


Journal ArticleDOI
TL;DR: Within the terminals, the synaptic organelles line up opposite periodic specializations in the myofiber's BL, demonstrating that components associated with the BL play a role in organizing the differentiation of the nerve terminal.
Abstract: Axons regenerate to reinnervate denervated skeletal muscle fibers precisely at original synaptic sites, and they differentiate into nerve terminals where they contact muscle fibers. The aim of this study was to determine the location of factors that influence the growth and differentiation of the regenerating axons. We damaged and denervated frog muscles, causing myofibers and nerve terminals to degenerate, and then irradiated the animals to prevent regeneration of myofibers. The sheath of basal lamina (BL) that surrounds each myofiber survives these treatments, and original synaptic sites on BL can be recognized by several histological criteria after nerve terminals and muscle cells have been completely removed. Axons regenerate into the region of damage within 2 wk. They contact surviving BL almost exclusively at original synaptic sites; thus, factors that guide the axon's growth are present at synaptic sites and stably maintained outside of the myofiber. Portions of axons that contact the BL acquire active zones and accumulations of synaptic vesicles; thus by morphological criteria they differentiate into nerve terminals even though their postsynaptic targets, the myofibers, are absent. Within the terminals, the synaptic organelles line up opposite periodic specializations in the myofiber's BL, demonstrating that components associated with the BL play a role in organizing the differentiation of the nerve terminal.

542 citations


Journal ArticleDOI
Zach W. Hall1
TL;DR: Although the activity of all three forms of acetylcholinesterase were decreased in denervated muscle, the largest proportional decrease occurred in theActivity of the 16 S form, which may correspond to the endplate enzyme.
Abstract: The properties of the cholinesterase activity in homogenates of whole rat diaphragm and of innervated (+EP) and non-innervated (−EP) regions of the muscle have been investigated. Under standard assay conditions, over 90% of the cholinesterase activity of whole muscle homogenates was due to specific acetylcholinesterases. The specific activity of acetylcholinesterase was higher in +EP regions of muscle than in −EP regions. About 40% of the total activity was calculated to be specifically associated with the endplates. When a high speed supernatant fraction of muscles homogenized in 1 M NaCl, 0.5% Triton X-100 was subjected to velocity sedimentation in a sucrose gradient, and three species of acetylcholinesterase activity with sedimentation constants of 4 S, 10 S and 16 S were observed. All three forms were stable under the conditions of sedimentation and had buoyant densities of approximately 1.28. All three hydrolyzed β-methylacetylcholine at approximately 30% the rate that acetylcholine was hydrolyzed. The 10 S and 16 S forms were inhibited by concentrations of acetylcholine over 1.25 mM, but no substrate inhibition was observed with the 4 S enzyme. Velocity sedimentation of extracts from +EP and −EP regions of muscle demonstrated that the 4 S and 10 S forms of the enzyme were distributed throughout the muscle while the 16 S form was found only in +EP regions. Extracts of the phrenic nerve contained only 4 S and 10 S forms. Thus, the 16 S form of acetylcholinesterase is specifically associated with endplate regions of muscle and may correspond to the endplate enzyme. Seven days after denervation of the diaphragm, both endplate-specific cholinesterase activity and the cholinesterase activity in −EP regions of muscle were decreased. Although the activity of all three forms of acetylcholinesterase were decreased in denervated muscle, the largest proportional decrease occurred in the activity of the 16 S form.

470 citations