Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Desensitization of acetylcholine (ACh) receptors was studied at the frog neuromuscular junction under voltage clamp.2. ACh was applied directly to junctional receptors by stimulating the motor nerve with trains of impulses. End-plate currents (e.p.c.s) were used to estimate the total number of channel openings by the junctional ACh receptors, and miniature end-plate currents (m.e.p.c.s) were used to measure changes in post-synaptic sensitivity. Under the conditions of these experiments the changes in m.e.p.c. amplitudes were shown to be post-synaptic in origin and thus provided a measure of desensitization.3. When the acetylcholinesterase was inhibited with diisopropylfluorophosphate, neostigmine, or collagenase treatment to prolong the duration of the nerve-released ACh in the synaptic cleft, desensitization developed during repetitive stimulation of 1000 impulses at 5-33 impulses/sec and then recovered after the conditioning trains, with a time constant of about 25 sec.4. When the acetylcholinesterase was active so that the duration of ACh in the synaptic cleft resulting from each nerve impulse was brief (< 300 musec), desensitization developed in response to 300-500 pairs of nerve stimuli if the interval between the impulses of each pair was 25 msec or less. When the interval was 30 msec or greater, however, measurable desensitization did not occur, even if the total number of channel openings was many times greater than in the experiments with shorter intervals or inhibited esterase where desensitization readily occurred.5. The desensitization observed to pairs of impulses was enhanced by chlorpromazine and decreased when the post-synaptic membrane was depolarized, properties similar to those described previously for desensitization to bath and ionophoretic application of ACh.6. These results indicate that desensitization to nerve-released transmitter is not a simple consequence of receptor activation, is not due to blockade of the open receptor channels by ACh, and does not result from ACh binding directly to desensitized receptors with a resulting shift in the receptor population towards the desensitized state.7. We suggest that the desensitization observed to nerve-released transmitter is a two-step process with both steps initiated by ACh. In the first step ACh converts some receptors into a desensitizable state which has an apparent lifetime of less than 30 msec; in the second step ACh desensitizes the desensitizable state.
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PMID:A study of desensitization of acetylcholine receptors using nerve-released transmitter in the frog. 627 65

The effects of beta-endorphin (lipotropin 61-91) and related naturally-occurring peptides upon acetylcholinesterase activity in rat hind-limb muscles was investigated. beta-endorphin weakly inhibited the activity in a plasma membrane-enriched fraction. The inhibition by beta-endorphin of the membrane-associated acetylcholinesterase was less marked when the fractions were prepared from muscles which had been denervated 4-6 days previously. The membrane-associated acetylcholinesterase was solubilised from normal muscle preparations and separated by sucrose density gradient centrifugation into three major peaks (16S, 10S and 4S). beta-Endorphin inhibited the activity in the 16S peak but not that in the 10S and 4S peaks, whilst tensilon, a competitive inhibitor of acetylcholinesterase, inhibited the activity of all three peaks. beta-Endorphin inhibited the activity in the 16S peak but not that in the 10S and 4S peaks, whilst tensilon, a competitive inhibitor of acetylcholinesterase, inhibited the activity of all three peaks. beta-Endorphin inhibited the 16S activity in a concentration-dependent manner and its action was partly prevented if naloxone was added simultaneously. Purified natural porcine and bovine beta-endorphin were equipotent in terms of effective concentration range but the maximum inhibition was greater with the bovine peptide. beta-Lipotropin was approximately 4 times less potent than beta-endorphin, whilst C-fragment (lipotropin 61-87) was 100 times less potent. Prolonged treatment with collagenase did not reduce the catalytic activity of 16S acetylcholinesterase, but it was no longer susceptible to the inhibitory action of beta-endorphin. Kinetic studies indicated a complex type of inhibition by beta-endorphin (hyperbolic Lineweaver-Burke plot). Methionine enkephalin inhibited acetylcholinesterase in a weakly non-competitive manner and its action was not abolished if the enzyme was predigested with collagenase. beta-Endorphin produces a novel form of inhibition of acetylcholinesterase, acting only on the 16S (A12 or 'motor endplate-specific') form of the enzyme. The findings are discussed in the light of evidence that beta-endorphin-related immunoreactivity is expressed in motor nerve axons in the immature rat.
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PMID:Selective inhibition of 'motor endplate-specific' acetylcholinesterase by beta-endorphin and related peptides. 628 23

Myotubes of a mouse muscle-cell line (C2) synthesize in culture a 16S form of acetylcholinesterase that is normally found only in regions of adult mouse muscle that contain endplates. The 16S enzyme in C2 cell extracts has the properties expected of acetylcholinesterase forms that have a collagen-like tail. In intact cells, the active site of the 16S acetylcholinesterase is protected by a membrane-impermeable inhibitor, and this form of the enzyme can be removed by treatment of the cells with collagenase. Thus the enzyme is extracellular. Its extraction by high ionic strength solutions lacking detergent suggests that the 16S form is associated with the extracellular matrix by ionic interactions. Histochemical staining shows focal patches of acetylcholinesterase activity on the cell surface. Collagenase treatment, which removes only the 16S form, abolishes this staining pattern, indicating that the patches consist of the 16S enzyme. We conclude that the 16S enzyme in C2 myotubes occurs in focal patches on the cell surface, where it is associated with the extracellular matrix.
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PMID:Association of the synaptic form of acetylcholinesterase with extracellular matrix in cultured mouse muscle cells. 628 45

The receptor for alpha-latrotoxin, the major protein component of the black widow spider venom, was investigated by the use of the purified toxin and of polyclonal, monospecific anti-alpha-latrotoxin antibodies. Experiments on rat brain synaptosomes (where the existence of alpha-latrotoxin receptors was known from previous studies) demonstrated that the toxin-receptor complex is made stable by glutaraldehyde fixation. At saturation, each such complex was found to bind on the average five antitoxin antibody molecules. In frog cutaneous pectoris muscles, the existence of a finite number of high-affinity receptors was revealed by binding experiments with 125I-alpha-latrotoxin (Kd = 5 X 10(-10) M; bmax = 1.36 +/- 0.16 [SE] X 10(9) sites/mg tissue, dry weight). Nonpermeabilized muscles were first treated with alpha-latrotoxin, and then washed, fixed, dissociated into individual fibers, and treated with anti-alpha-latrotoxin antibodies and finally with rhodamine-conjugated sheep anti-rabbit antibodies. In these preparations, muscle fibers and unmyelinated preterminal nerve branches were consistently negative, whereas bright specific fluorescent images, indicative of concentrated alpha-latrotoxin binding sites, appeared in the junctional region. These images closely correspond in size, shape, and localization to endplates decorated by the acetylcholinesterase reaction. The presynaptic localization of the specific fluorescence found at frog neuromuscular junctions is supported by two sets of findings: (a) fluorescent endplate images were not seen in muscles that had been denervated; and (b) the distribution of fluorescence in many fibers treated with alpha-latrotoxin at room temperature was the one expected from swollen terminal branches. Swelling of terminals is a known morphological change induced by alpha-latrotoxin in this preparation. When muscles were treated with either proteolytic enzymes (trypsin, collagenase) or detergents (Triton X-100) before exposure to alpha-latrotoxin, the specific fluorescent endplate images failed to appear. Taken together these findings indicate that the alpha-latrotoxin receptor is an externally exposed protein highly concentrated in the nerve terminal plasma membrane. Its density (number per unit area) at the frog neuromuscular junction can be calculated to be approximately 2,400/micron2.
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PMID:Specific localization of the alpha-latrotoxin receptor in the nerve terminal plasma membrane. 633 Jan 24

Electrophorus electricus acetylcholinesterase is a large polymorphic enzyme. Its native forms 18 S, 14 S and 8.5 S possess a tail having a collagen-like structure. It was suggested that this tail is involved in the anchorage of the enzyme at the terminal of the synapse. Watkins et al. [1] showed that all forms of the enzyme having a collagen segment also bind to sphingomyelin liposomes with almost no binding to phosphatidylcholine (PC) liposomes. In agreement with the above results, the binding of acetylcholinesterase reported here was independent of the following liposomal parameters (a) curvature, (b) the physical state of the bilayer, (c) the gel to liquid crystalline phase transition of sphingomyelin, (d) stereospecificity of the sphingomyelin, (e) acyl chain of the sphingomyelin. The binding was reduced with increasing PC content in sphingomyelin vesicles. The binding has no effect on the bilayer integrity. The enzymatic activity can be released from the vesicles by incubation with collagenase. The association of the enzyme with the liposomes had minimal effect on its kinetic parameters (Km, Vmax). The only detectable effect was increasing enzyme stability at low enzyme concentration. This suggested that the binding of the enzyme to sphingomyelin liposomes reduced its surface denaturation. Such association was not unique to acetylcholinesterase since collagen showed similar behavior. Collagen binding to sphingomyelin liposomes was 5-10-times larger than to PC liposomes. The exact details of the interaction of collagen and collagen-like peptides with sphingomyelin bilayers are yet unknown although it differs from the well documented hydrophobic or electrostatic interactions [7]. This work proposes hydrogen bonding as a third mechanism which involves the interface region of sphingolipids molecules and the collagen or collagen-like tail of acetylcholinesterase. This binding is also of interest due to its correlation to the accumulation of sphingomyelin and collagen during aging and the development of atherosclerosis in blood vessels of mammals.
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PMID:Characterization of the association of Electrophorus electricus acetylcholinesterase with sphingomyelin liposomes. Relevance to collagen-sphingomyelin interactions. 649 89

The characterization of individual acetylcholinesterase (AChE) molecular form subcellular pools in adult mammalian skeletal muscle is a critical point when considering such questions as the origin, assembly, and neurotrophic regulation of these molecules. By correlating the results of differential extraction, in vitro collagenase digestion, and in situ pharmacologic probes of AChE molecular forms in endplate regions of adult rat anterior gracilis muscle, we have shown that: 1) 4.0S (G1) and 6.0S (G2) AChE are predominantly membrane-bound and intracellular; if an extracellular and/or soluble fraction of these forms exists, it cannot be adequately resolved by our methods; 2) 9-11S (globular) AChE activity is distributed between internal and external pools, as well as membrane-associated and soluble fractions; 3) 16.0S (A12) AChE is not an integral membrane protein and exists both intracellularly (25-30%) and extracellularly (70-75%).
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PMID:Subcellular localization of acetylcholinesterase molecular forms in endplate regions of adult mammalian skeletal muscle. 650 36

The cholinesterase activity of Xenopus laevis oocytes was assessed using [3H]acetylcholine in a simple radiometric procedure. The cholinesterase activity of mature (stage V-Vl) oocytes was very sensitive to inhibition by the specific acetylcholinesterase inhibitor, BW284-C5l, and relatively insensitive to an inhibitor of non-specific, or butyrylcholinesterase. The Km and Vmax of the acetylcholinesterase measured in homogenates of oocytes were 312 microM and 4.6 nmol-oocyte 1-h 1, respectively. Triton X-100 increased the enzyme activity of homogenates four- to five-fold while collagenase treatment displaced into the medium none of the acetylcholinesterase activity from either homogenates or intact oocytes. Cations were found generally to diminish the acetylcholinesterase activity of oocyte homogenates, and lanthanum ions inhibited acetylcholine hydrolysis with an IC50 of 0.63 mM. Subcellular fractionation of oocytes revealed that the bulk of enzyme activity was associated with particulate fractions. Acetylcholinesterase activity was also detected on the surface, and in homogenates, of immature oocytes. Peak enzyme activity resided in stage IV oocytes. Eggs obtained from females induced to spawn were found to have acetylcholinesterase activity in homogenates but little or no hydrolytic activity was detected on the egg surface. These results provide a point of departure for further investigations of the functional significance of this enzyme in Xenopus oocytes.
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PMID:Acetylcholinesterase activity of Xenopus laevis oocytes. 666 98

We have distinguished three fractions of acetylcholinesterase (AcChoE; acetylcholine acetylhydrolase, EC 3.1.1.7) from Torpedo marmorata electric organs, according to their solubilization characteristics. The low-salt-aggregating collagen-tailed forms are soluble in high-salt buffers; their hydrodynamic properties ae not modified in the presence of detergents. They constitute the A fraction, which amounts to about a third of the tissue's AcChoE activity. The low-salt-soluble (LSS) and detergent-soluble (DS) fractions are not sensitive to ionic strength and collagenase. In the presence of nonionic detergents or bile salts, both fractions behave as a monodisperse "6.3S" form, the properties of which have been investigated mostly in the case of Triton X-100. Disulfide bond reduction dissociates the detergent form into a smaller "5S" form. These two forms are thought to be, respectively, detergent-associated dimers and monomers. In the absence of detergent, the LSS fraction is polydisperse: it contains a major 8S component, 11S and 14S components, and faster-sedimenting aggregates, which appear to represent dimers, tetramers, and higher polymers. The heterogeneity of the 8S component in gel filtration suggests that it also contains variable noncatalytic elements. Upon removal of the detergent the DS fraction forms ill-defined aggregates. Trypsin induces quaternary rearrangements of part of the 8S component into 11S and 14S components, which are still convertible into the detergent form; therefore trypsin probably digests noncatalytic elements. Pronase and proteinase K, on the other hand, convert the enzyme into a dimeric form, G2, that does not interact with detergents, probably by cleaving a minor fragment of the subunit that is involved in hydrophobic interactions.
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PMID:Collagen-tailed and hydrophobic components of acetylcholinesterase in Torpedo marmorata electric organ. 693 97

The levels and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and pseudocholinesterase (psiChE, EC 3.1.1.8) were examined in various skeletal muscles, cardiac muscles, and neural tissues from normal and dystrophic chickens. The relative amount of the heavy (Hc) form of AChE in mixed-fibre-type twitch muscles varies in proportion to the percentage of glycolytic fast-twitch fibres. Conversely, muscles with higher levels of oxidative fibres (i.e., slow-tonic oxidative-glycolytic fast-twitch, or oxidative slow-twitch) have higher proportions of the light (L) form of AChE. The effects of dystrophy on AChE and psiChE are more severe in muscles richer in glycolytic fast-twitch fibres (e.g., pectoral or posterior latissimus dorsi, PLD); there is no alteration of AChE or psiChE in a slow-tonic muscle. In the pectoral of PLD muscles from older dystrophic chickens, however, the AChE forms revert to a normal distribution while the pesChE pattern remains abnormal. Muscle psiChE is sensitive to collagenase in a similar way as is AChE, thus apparently having a similar tailed structure. Unlike skeletal muscle, cardiac muscle has very high levels of psiChE, present mainly as the L form; AChE is present mainly as the medium (M) form, with smaller amounts of L and Hc. The latter pattern of AChE forms resembles that seen in several neural tissues examined. No alterations in AChE or psiChE were found in cardiac or neural tissues from dystrophic chickens.
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PMID:Comparison of the molecular forms of the cholinesterases in tissues of normal and dystrophic chickens. 706 26

The developmental profiles of the enzyme acetylcholinesterase, and of some of its quaternary structural forms, characterized by discrete sedimentation coefficients, have been comparatively analyzed in chick retina and optic tectum, between embryonic day 8 and day 10 after hatching. Four molecular species of AChE have been characterized in both retina and tectum during this developmental period: two of them with sedimentation coefficients of 11S and 6S, accounting together for 94-99% of the AChE activity in the initial homogenate, can be easily extracted by homogenization in a buffer containing 1% Triton X-100 and 1 M NaC1, at 4 degrees C. The other two, however, are not extractable by such treatment, but can be released by collagenase from the residue left after the detergent-salt extraction; they have apparent sedimentation coefficients of 21.5S and 16.5S and represent, together, less than 2% of activity in the initial homogenate. All four forms of the enzyme show distinctive patterns of change during the developmental period considered, with significant differences between retina and tectum. These differences are discussed in the context of the specific roles of retina and tectum in the visual process.
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PMID:Molecular forms of acetylcholinesterase in the developing chick visual system. 721 2


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