Interaction of Asymmetric and Globular Acetylcholinesterase Species with Glycosaminoglycans

Abstract

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)

Chondroitinases release acetylcholinesterase from chick skeletal muscle

“…We have checked three different heparinases taking into account the structural heterogeneity of the heparin/heparan sulfate family of GAGS: it is, for instance, a proven fact that heparan sulfates do often contain long heparin-like stretches [ 121. Besides, in our hands, heparin shows consistently more affinity for tailed AChE forms than heparan sulfate itself [8,9]. Actually, these enzymes show mixed specificities: 'heparinase' (heparinases I and II) acts equally well upon heparin and heparitin sulfaies C and D, wherecs heparitinase I (heparinase III) degra Ies only heparitin sulfates A and B [13].…”

“…To explore, within the latter hypothesis, the possibility of a joint involvement of both heparan and chondroitin sulfates in the attachment of extracellular muscle AChE we also measured the release of A-forms by a mixture of all heparinases Ten-day chick muscle samples, preextracted with TT buffer, were incubated with 5 U/ml of chondroitinase, as described in section 2, along with control samples (background -Bckg -control, containing no enzyme, and maximal release control -valid only for A-forms -, containing high salt and EDTA). The digested samples were usually freed of the coarse particulate material by filtration, and then layered on top of a S-2O"Io sucrose gradient, made up in TTSE buffer, together with sedimentation markers [8]. These gradients were run, fractionated and assayed for ACM2 (nmol ['"CJacetate released per min and fraction) and marker enzyme activities as described IB].…”

“…Among glycosaminoglycans (GAGS), heparan sulfate has been singled out as the most likely candidate for this role in view of the proven affinity of the tailed AChE species for heparin [3][4][5][6]8], and of the reported ability of heparinases (but not chondroitinases) to dissociate aggregative AChE-GAG complexes [2], and to release asymmetric AChE from electric organ samples [S]. Our recent work has shown, however, that the affinity of heparin for the asymmetric AChE tail is not a selective phenomenon, being shared by a number of polyanions including sulfated GAGS, dextran sulfates, some acidic polyaminoacids and even polyvinylsulfates [9].…”

Acetylcholinesterase from Schistosoma mansoni: interaction of globular species with heparin

“…This conclusion is based on the following findings : (1) The bulk of the enzyme is extracted at low ionic strength, in the presence of detergent ; (2) sedimentation on sucrose gradients is not affected by collagenase ; and (3) the close migration of the 6.5 S and 8 S species on gel filtration ( Figure 5) argues against the 8 S species being an A % form, rather than a G form. Although this assignment is unusual, interaction of G forms of both AChE and butyrylcholinesterase with heparin has been reported [22,23].…”

“…The G forms of AChE lack the collagen tail, and are usually considered to lack the capacity to interact with heparin. However, Ramirez et al [22] showed that the G % form from chick muscle binds to heparin with low affinity. Furthermore, Sine et al [23] reported an interaction with heparin by an amphiphilic G # form of butyrylcholinesterase extracted from mucosal cells of rat intestine.…”