Solubilization of collagen-tailed molecular forms of acetylcholinesterase from several brain areas in different vertebrate species.

The relative efficiency of a buffered medium containing a high salt concentration and EDTA as a means to solubilize collagen-tailed molecular forms of acetylcholinesterase has been examined in four brain areas of several species belonging to different vertebrate classes. This extraction procedure has proved successful in most cases, with the yield of tailed enzyme varying between less than 1 and 26% of the total tissue activity. The solubilization values are consistently higher in more primitive vertebrates than in mammals and, for a given species, are usually lower in the telencephalon than in other brain structures. Our results confirm that the vertebrate central nervous system contains collagen-tailed quaternary structural forms of acetylcholinesterase.

Collagen-tailed and globular forms of acetylcholinesterase in the developing chick visual system.

We have carried out a comparative study of the developmental profiles of the enzyme acetylcholinesterase, and of its collagen-tailed and globular structural forms, solubilized in the presence of 1 M NaCl, 1% (w/v) sodium cholate and 2 mM EDTA, in the chick retina and optic lobes. The overall acetylcholinesterase activities, both per mg protein and per embryo or chick, are substantially higher in tectum than in retina, from embryonic day 16. The A(12) collagen-tailed form of the enzyme is present in similar amounts in the embryonic retina and optic tectum; however, while the A(12) activity increases significantly in retina after birth, both by percentage and in absolute terms, the tectal tailed enzyme follows a declining developmental profile, reaching a minimum after 6 months of life. On the other hand, the globular G(4) species shows developmental profiles, both in retina and tectum, rather similar to those obtained for the overall enzyme activity, while the G(2) and G(1) forms are present in comparable concentrations in both tissues. Besides, G(4) is the predominant globular form in the chick optic lobe after hatching, G(2) and G(1) being enriched in the embryonic tectum. In the case of retina, however, all the globular forms contribute more evenly to the total acetylcholinesterase activity, along the developmental period considered. The potential significance of some of the postnatal developmental profiles is discussed in terms of the progressive adjustment of retina and tectum to the requirements of visual function.

Fate of collagen-tailed acetylcholinesterase upon extraction of chick retina with salt-detergent solutions in the absence of EDTA.

In chick retina, the tailed 20S molecular form of acetylcholinesterase (A12) is slowly degraded to globular forms after homogenization of the tissue in a buffer-salt-detergent solution, in the absence of EDTA. This process can be stopped by the addition of EDTA to the homogenate, prior to high speed centrifugation; however, longer delays in adding EDTA lead to lower recoveries of tailed enzyme. The rate of degradation of A12 is considerably enhanced by high speed centrifugation of this EDTA-less homogenate prior to addition of EDTA.

Solubilization of collagen-tailed acetylcholinesterase from chick retina: effect of different extraction procedures.

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.