Heparin-acetylcholinesterase interaction: Specific detachment of class I-A forms and binding of class I and II-A forms to heparin-agarose (original) (raw)
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Heparin solubilizes asymmetric acetylcholinesterase from rat neuromuscular junction
FEBS Letters, 1983
We are interested in the factors involved in the anchorage of acetylcholinesterase (AChE) to the synaptic basal lamina. Here, we report studies showing that heparin, a sulfated glycosaminoglycan, specifically solubilized AChE from endplate regions of rat diaphragm muscle. Of the several molecular forms of AChE present in that region, heparin only released the asymmetric Al2 and As forms of the enzyme. Our results strongly support the involvement of heparin-like macromolecules in the in vivo immobilization of the collagen-tailed forms of AChE to the basal lamina of the neuromuscular junction.
Binding of the asymmetric forms of acetylcholinesterase to heparin
Biochemical Journal, 1984
The interaction between acetylcholinesterase (EC 3.1.1.7) and heparin, a sulphated glycosaminoglycan, was studied by affinity chromatography. A specific binding of the asymmetric acetylcholinesterase to an agarose gel containing covalently bound heparin was demonstrated. This interaction required an intact collagenous tail, shown by the fact that the binding is abolished by pretreatment with collagenase. The globular forms did not bind to the column. Both total and intracellular asymmetric acetylcholinesterase forms isolated from the endplate region of the rat diaphragm muscle showed higher affinity for the heparin than did the enzyme from the non-endplate region. The binding to the resin was destabilized with 0.55 M-NaCl, and, among the various glycosaminoglycans tested, only heparin was able to displace the acetylcholinesterase bound to the column. Our results added further support to the concept that the asymmetric acetylcholinesterase forms are immobilized on the synaptic basal ...
Brain Research, 1990
We are interested in the study of the interactions involved in the attachment of collagen-tailed acetylcholinesterase (ACHE) to the synaptic basal lamina. The fact that AChE occupies less than 0.1% of the muscle basal lamina, suggests that there is a very high specificity in the interaction that defines its distribution. We have previously found that asymmetric AChE is bound to the neuromuscular junction via heparan sulfate proteoglycans. Sulfated glycosaminoglycans as heparan sulfate and heparin extracted the asymmetric AChE from the synaptic basal lamina. Here we show that dermatan sulfate as well as de-sulfated heparin, are also able to extract collagen-tailed ACHE. Taking into account that the sohibilization of the asymmetric AChE is concomitant with the liberation of a dermatan sulfate proteoglycan from the rat neuromuscular junction, the present results open the possibility that the collagen-tailed AChE is also anchored to dermatan sulfate proteoglycans at the synaptic basal lamina. Surgical procedures Male Sprague-Dawley rats of 250-300 g were maintained with free access to food and water. Their diaphragms were removed from the ribs, washed and kept in ice-cold saline. The muscles were then divided into endplate and non-endplate regions t3. Only the endplate region were used in this work. AChE extraction procedure The experiments were performed using sequential extraction procedures. The tissue was weighed, finely minced with scissors and homogenized (1:10 w/v) in low ionic strength buffer, 25 mM
Interaction of Asymmetric and Globular Acetylcholinesterase Species with Glycosaminoglycans
Journal of Neurochemistry, 1990
Chicken muscle and retina, and rat muscle asymmetric acetylcholinesterase (AChE) species were bound to immobilized heparin at 0.4 M NaCI. Binding efficiency was between 50 and 80% for crude fraction I A-forms (A'; muscle), and nearly 100% for fraction I1 A-forms (A"; muscle and retina). Antibody-affinity-purified A'-forms (chicken) were, however, quantitatively bound to heparin-agarose gels, whereas diisopropylfluorophosphate-inactivated high-salt extracts partially prevented the binding of both A' and A" AChE forms, thus suggesting the presence in crude A' 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 hcparin in the absence of salt. Stepwise elution with increasing NaCI concentrations showed maximal release of G-forms at 0.15 M, all
The Journal of Cell Biology
Heparan sulfate and heparin, two sulfated glycosaminoglycans (GAGs), extracted collagen-tailed acetylcholinesterase (ACHE) from the extracellular matrix (ECM) of the electric organ of Discopyge tschudii. The effect of heparan sulfate and heparin was abolished by protamine; other GAGs could not extract the esterase. The solubilization of the asymmetric AChE apparently occurs through the formation of a soluble AChE-GAG complex of 30S. Heparitinase treatment but not chondroitinase ABC treatment of the ECM released asymmetric AChE forms. This provides direct evidence for the in vivo interaction between asymmetric AChE and heparan sulfate residues of the ECM. Biochemical analysis of the electric organ ECM showed that sulfated GAGs bound to proteoglycans account for 5% of the total basal lamina. Approximately 20% of the total GAGs were susceptible to heparitinase or nitrous acid oxidation which degrades specifically heparan sulfates, and ~80% were susceptible to digestion with chondroitinase ABC, which degrades chondroitin-4 and -6 sulfates and dermatan sulfate. Our experiments provide evidence that asymmetric AChE and carbohydrate components of proteoglycans are associated in the ECM; they also indicate that a heparan sulfate proteoglycan is involved in the anchorage of the collagen-tailed AChE to the synaptic basal lamina.
FEBS Letters, 1987
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 macromol~ules 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. Acetylcholinesterase; Heparin; Dermatan sulfate proteoglycan; (Muscle extracellular matrix) Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/87/%3.50 0 1987 Federation of European Biochemical Societies 159
Journal of Biological Chemistry, 2003
ColQ, the collagen tail subunit of asymmetric acetylcholinesterase, is responsible for anchoring the enzyme at the vertebrate synaptic basal lamina by interacting with heparan sulfate proteoglycans. To get insights about this function, the interaction of ColQ with heparin was analyzed. For this, heparin affinity chromatography of the complete oligomeric enzyme carrying different mutations in ColQ was performed. Results demonstrate that only the two predicted heparin-binding domains present in the collagen domain of ColQ are responsible for heparin interaction. Despite their similarity in basic charge distribution, each heparin-binding domain had different affinity for heparin. This difference is not solely determined by the number or nature of the basic residues conforming each site, but rather depends critically on local structural features of the triple helix, which can be influenced even by distant regions within ColQ. Thus, ColQ possesses two heparinbinding domains with different properties that may have non-redundant functions. We hypothesize that these binding sites coordinate acetylcholinesterase positioning within the organized architecture of the neuromuscular junction basal lamina.
Collagenase-induced alteration in mouse 16S acetylcholinesterase
Brain Research, 1979
Acetylcholinesterase AChE (EC 3,1,1,7) is present in high concentration at the vertebrate neuromuscular junction where it catalyses the hydrolysis of neuronallyreleased acetylcholine7,2L When rat striated muscle is extracted in a medium containing non-ionic detergents, the AChE activity can be resolved into three major distinct forms with sedimentation coefficients of 4S, 10S and 16S by sucrose gradient sedimentation. The 16S form has been shown to be restricted to the motor end-plate region and to be present only when motor innervation is intactl°,Zl, 23. In his initial studies on the 16S form, Hall TM demonstrated that it was selectively released into the medium by treatment of the neuromuscular junction with crude collagenase. Other studies have shown the release of AChE activity or the diminution of residual AChE activity by proteolytic enzymes1, 3 and by the use of collagenase preparations free of tryptic activity 4. Similar release of activity by purified collagenase has been seen in the electric fish 14. Unfortunately, these studies do not answer the question of whether the collagenase acts on an AChE 'attachment site' in the basal lamina lz or on a collagenasesensitive structure intrinsic to the 16S AChE molecule. In the case of mammalian AChE a definitive answer is made more difficult since biochemical quantities of the various molecular forms are not available. Therefore, we have decided on an indirect approach by studying the sedimentation and aggregation properties of AChE obtained from muscle by detergent extraction followed by coilagenase treatment. In these studies, 2-month-old C57/BL6/J mice were decapitated and immediately dissected. The gastrocnemius muscles were put on ice, weighed and then homogenized in a conical glass homogenizer in 10-2 M Tris buffer at pH 7.2 containing 1 M NaCI, 0.05 M MgCI2 and l ~ Triton X-100 in proportions of one part muscle to 10 parts buffer (w/v). The homogenate was centrifuged at 30,000 × g for 15 min in a Sorvall RC-2B centrifuge. The supernatants were collected and incubated with 250 units of pure, reconstituted collagenase, form III (Advanced Biofactures Corp., Lynnbrook, N.Y.
Journal of Biological Chemistry, 1998
Collagen-tailed asymmetric acetylcholinesterase (AChE) forms are believed to be anchored to the synaptic basal lamina via electrostatic interactions involving proteoglycans. However, it was recently found that in avian and rat muscles, high ionic strength or polyanionic buffers could not detach AChE from cell-surface clusters and that these buffers solubilized intracellular non-junctional asymmetric AChE rather than synaptic forms of the enzyme. In the present study, asymmetric AChE forms were specifically solubilized by ionic buffers from synaptic basal lamina-enriched fractions, largely devoid of intracellular material, obtained from the electric organ of Torpedo californica and the end plate regions of rat diaphragm muscle. Furthermore, foci of AChE activity were seen to diminish in size, number, and staining intensity when the rat synaptic basal lamina-enriched preparations were treated with the extraction buffers. In the case of Torpedo, almost all the AChE activity was removed from the pure basal lamina sheets. We therefore conclude that a major portion of extracellular collagen-tailed AChE is extractable from rat and Torpedo synaptic basal lamina by high ionic strength and heparin buffers, although some non-extractable AChE activity remains associated with the junctional regions.