PubMed · 55983
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V P Babmindra, T A Bragina. 1975. [Several problems in current synaptology].. https://pubmed.ncbi.nlm.nih.gov/55983/
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We studied survival and expression of cholinergic properties in embryonic septal neurons grown in very low density microcultures (1-7 cells per Terasaki well). Even in cultures containing only a single neuron, at least 10% of plated neurons survived for 2 weeks or more in medium containing fetal calf serum or an acid-stable fraction (55,000 Da) of horse serum. Of these surviving neurons, 30-40% stained positively for acetylcholinesterase (AChE) or nerve growth factor (NGF) receptor, even though the culture medium lacked detectable levels of NGF, brain-derived neurotrophic factor, and fibroblast growth factor. Addition of NGF or an antibody against NGF had no effect on either neuronal survival or the percentage of neurons staining positively for AChE or NGF receptor after 18-20 days in vitro. There was no cell division in medium containing the serum fraction, but when 10% fetal calf serum was present cell division occurred in some of the cultures, and in half of these cases at least one of the clonal progeny became AChE-positive. These results demonstrate that some embryonic septal cells can survive at least 2 weeks and develop cholinergic neuronal properties in the absence of other cells or NGF.
Cell-bound acetylcholinesterase (AChE) was found to be an early differentiation marker on embryonic chick skeletal myoblasts in mixed primary cell cultures. AChE biosynthesis was detected and characterized by (a) a sensitive microtiter assay, (b) use of selective inhibitors, and (c) with mono- and polyclonal antibodies. Both secreted and cell-bound AChE appeared on the first day in culture, at a time when no muscle cell fusion was observed. Characterization of this enzyme revealed that true AChE was bound and secreted by myoblasts. BW284c51, which permeates cell membranes poorly, inhibited all the cell-associated AChE activity on myoblasts, suggesting that the activity measured was on the outer cell surface. On the other hand, fibroblasts appeared to have no or very little bound enzyme and the low level of secreted enzyme activity had the characteristics of pseudo-, or butyrylcholinesterase. Polyclonal anti-Torpedo californica electroplax AChE antibody and several monoclonal antibodies were found to bind specifically to chick myoblasts. Since the cells had not been made permeable before antibody binding, a membrane-bound form of the enzyme was most likely being detected. The cell-bound true AChE was present in identifiable quantities from the first day of culture. Membrane-bound AChE can thus serve as an early differentiation marker for embryonic chick myoblasts in mixed primary cultures.
Modification of Torpedo californica acetylcholinesterase (AChE) both by bis(1-oxy-2,2,5,5-tetramethyl-3-imidazolin-4-yl)disulfide (biradical) and by 4,4'-dithiopyridine, via a thiol-disulfide exchange reaction, was monitored by EPR and optical spectroscopy, respectively. Incubation with these reagents caused complete loss of enzymic activity. Treatment with glutathione of AChE modified by either of the two disulfides led to rapid release of the bound reagent with simultaneous regeneration of the single free thiol group of the enzyme. However, no concomitant recovery of catalytic activity was observed. SDS-PAGE showed that both the modified and demodified enzymes retained their structure as a disulfide-linked dimer. Circular dichroism revealed that modification of AChE by the disulfide agents with or without demodification by glutathione led to a complete disappearance of the ellipticity in the near-UV and to a much smaller decrease in ellipticity in the far-UV. The CD spectra observed are typical of the "molten globule" state of proteins. 1-Anilino-8-naphthalenesulfonate binding measurements and an enhanced susceptibility to trypsinolysis supported the supposition that chemical modification had transformed native AChE to a "molten globule".