Solubilization of Collagen‐Tailed Acetylcholinesterase from Chick Retina: Effect of Different Extraction Procedures

Abstract

We have extracted acetylcholinesterase from young chick retinas by homogenization in different solutions combining high salt concentration, ionic and nonionic detergents, and EDTA, looking for an optimum procedure for the solubilization of collagen-tailed, asymmetric structural forms of the enzyme. High salt and EDTA seem to be the only necessary requirements for the solubilization of acetylcholinesterase as the A12 form (20S), and the presence of detergent in the homogenization medium does not significantly improve the yield of tailed enzyme. Extraction in the absence of detergent has the potential advantage of a threefold enrichment of tailed enzyme, because only about one-third of the total retinal acetylcholinesterase activity is solubilized. Divalent cations, especially Ca2+, seem to be involved in the attachment of the tailed enzyme to the retinal membranes, at the tail level. High salt-EDTA-extracted 20S acetylcholinesterase (without detergent) aggregates in the presence of exogenous Ca2+ and becomes "insoluble." However, the aggregated 20S acetylcholinesterase can be completely recovered and brought back into solution by further addition of EDTA. Besides, the aggregation can be prevented by the inclusion of Triton X-100 in the homogenization buffer or by adding the detergent concurrently with Ca2+. It is postulated that the acetylcholinesterase collagenous tail is coated by acidic lipid molecules hydrophobically bound to the tail protein so that Ca2+ ionic bridges would actually link these lipid molecules (and consequently the tail) to the membrane matrix. Removal of the lipid coat (e.g., by Triton X-100) produces tailed acetylcholinesterase molecules that no longer aggregate in the presence of Ca2+ and are fully accessible to collagenase digestion.

Specific binding of [3H]GppNHp to extracellular membrane receptors in chick cerebellum: possible involvement of kainic acid receptors

“…We have previously characterized KA binding sites in chick tectal and cerebellar membranes from pharmacological and developmental standpoints [16, 17]. We have also shown that GNs, including GMP, are efficient displacers of [ 3 H]KA [8, 11].…”

“…Results with chick KA receptors are specially illustrative in this respect [8, 10, 11]. Chick tectal or cerebellar membranes contain abundant medium/low affinity binding sites ( K d in the range of 100–300 nM) that have been analyzed in different laboratories [14, 16, 17]and, in more recent years, purified and cloned [14, 18].…”

Modes of attachment of acetylcholinesterase to the surface membrane

“…They were, however, subsequently observed in the motor nerve itself [19,75,761, in various peripheral ganglia [23, 771 and, subsequently, in the central nervous system. Thus Ramirez and coworkers [78,79] clearly demonstrated the presence, in the chick visual system (i.e. in tectum and retina) of A forms displaying the same physicochemical parameters and collagenase sensitivity as the corresponding forms in chick skeletal muscle.…”

Guanine nucleotides protect against kainate toxicity in an ex vivo chick retinal preparation

“…1). Thus retina differs from other chick brain regions, such as tectum and cerebellum, where only the low‐affinity site has been found [10, 25]. These receptors would be, in principle, the molecular substrate for the excitotoxic action of KA in the chick retina, already a firmly established fact [14–21, 26, 27].…”