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Title: Studies on neuronal proteins
Author: Chubb, I. W.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1975
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Abstract:
This thesis is concerned with studies on acetylcholinesterase. The problem of the role and fate of the protein has been studied using a variety of techniques. 1. Biochemical experiments. When homogenates, in isotonic media (0.3M sucrose), of bovine splanchnic axons and adrenal medullae were subfractionated in a centrifuge, it was found that the acetylcholinesterase did not give the expected distribution. Much of the early work in this field had resulted in the conclusion that acetylcholinesterase was a uniquely membrane-bound enzyme (see e.g. Boell and Nachmansohn, 1940: Toschi, 1959) but my results suggested that a considerable proportion of the activity in both tissues was soluble. Thus, 47% of the acetylcholinesterase activity present in the splanchnic axons and 31% of the enzyme in the adrenal medullae could, not be sedimented by prolonged high-speed centrifugation. The possibility that the preparation of the tissue for the centrifugation experiments might have "solubilized" the acetylcholinesterase from its normal, membrane-bound localization was investigated in several ways. It was found: that different strengths of homogenization, while having the expected effect of breaking up the tissue into smaller pieces, had no effect on the proportion of the enzyme which was not sedimentable; that suspension and "homogenization" of the membranes themselves did not result in a solubilization of their acetylcholinesterase; that incubation, at 25°, of a washed fraction containing only sedimentable acetylcholinesterase activity did not result in any solubilization of the enzyme. It was concluded that the soluble enzyme was not artifactually derived from the membrane-bound form during the preparation of the tissues for the earlier experiments. Because acetylcholinesterase is an enzyme which will hydrolyze a variety of substrates, it can be localized by histochemical methods. In addition to using this technique on tissues (see below) the method has also been used to locate the enzyme on polyacrylamide gels after different tissue extracts had been subjected to electrophoresis. The results of this type of analysis showed that the acetylcholinesterase of the two tissues was separable into several isoenzymes (see Webb, 1964 for definition of term isoenzyme). The simplest case was found in splanchnic nerve axons, where there was only one membrane-bound form of the enzyme (revealed by an extraction of the enzyme activity with Triton X-100 prior to electrophoresis) and another, different, form in the high-speed supernatant. The membrane-bound isoenzyme WRS always unique but occasionally there was more than one soluble isoenzyme. However, even in these rare cases, it was qualitatively estimated that more than 95% of the total activity on the gel was due to the one normal isoenzyme. The adrenal medullae presented a more complex picture. Again, there was only one membrane-bound isoenzyme, which had the same electrophoretic mobility as the axonal form, but there was a greater number of soluble forms of the acetylcholinesterase. One of these appeared to be the same form as was soluble in the axons, but in addition there were usually four others unique to the medulla. These latter isoenzymes had higher electrophoretic mobilities than either of the two isoenzymes of the splanchnic nerve. The different isoenzymes were numbered from 1, the fastest migrating, to 6, the slowest. Thus, AChE6 and AChE5 represent, respectively, the membrane-bound and the slowest migrating of the soluble isoenzymes. These are the two forms which are common to both axons and medullae. The relationship between AChE5 and AChE6, was investigated in two ways. Electrophoresis over a range of polyacrylamide gel concentrations showed that they each changed their mobility in an identical fashion, i.e., a plot of log10(Rm andtimes; 100) v. polyacrylamide concentration yielded two essentially parallel lines. This, according to Hedrick and Smith (1968), indicates that the two isoenzymes have very similar molecular sizes but different electrical charges. Supporting evidence for this suggestion was obtained by centrifugation of the acetylcholinesterase on stabilizing sucrose gradients; both the soluble and, after extraction with Triton X-100, the membrane-bound acetylcholinesterase sedimented to the same area of the gradient. Thus, when it was found that AChE5 behaves as though it has a molecular weight of 240,000, this also indicated a similar size for AChE6. Experiments of this type were also used to analyze the relationship between the soluble isoenzymes of the adrenal medulla. These results showed that whereas medullary AChE5 was identical to the soluble isoenzyme in the axons, the faster migrating forms all differed from it in size at least, and possibly also in charge. Because of the ease with which AChE5 and AChE6 can be separated, a few properties of each were compared without prior and extensive purification of the proteins. It was found that they had an identical apparent Km (7.5 andtimes; 10-5M, acetylthiocholine as substrate), identical Q10's of 1.2 and identical heat denaturation properties. Thus, the only differences between them were in their relative solubilities and their electrophoretic mobilities. To try to distinguish whether the soluble isoenzyme in the axons was contained within the cytoplasm, or whether it was within a labile particle which homogenization broke to liberate the soluble internal constituents, we investigated the rate at which acetylcholinesterase accumulated in constricted splanchnic axons and analyzed the types of isoenzyme involved in the movement. Both AChE5 and AChE6. were found to accumulate at an identical, relatively rapid rate in these nerves. It was concluded that the AChE5, and at least some of the AChE6, was contained within a fragile particle and that the soluble enzyme was liberated from this particle during the homogenization procedure. 2. Studies on the release of acetylcholinesterase from the isolated adrenal gland. A rapid flow rate of the acetylcholinesterase is a strong indication that there is an equally rapid removal of the enzyme from, presumably, the nerve endings. One way in which such a rapid removal could take place is by a release of the protein from the nerve. To investigate this possibility, we stimulated the isolated, perfused bovine adrenal gland with a variety of secretagogues to try and induce release of the acetylcholinesterase. It was found that the enzyme could be released by administering either a high concentration of K+, Dimethylphenylpiperazinium Iodide or Carbachol to the gland. The release was dependent upon the presence of Ca2+ ions in the perfusing fluid. Electrophoretic analysis of the perfusate showed that only AChE5 was released from the glands. The amount of enzyme appearing in the perfusate was related to the amount of catecholamines, probably reflecting the efficacy of the stimulation, but not to the amount of acetylcholinesterase in the adrenal gland. 3. Cytochemical studies. Because only one of several soluble isoenzymes of acetylcholinesterase was released, and since we were unable to analyze the storage characteristics of the protein by the normal methods (centrifugation) we resorted to a cytochemical analysis of the bovine adrenal medullae and splanchnic axons in an attempt to find how, and in what structure, the acetylcholinesterase was stored.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.451489  DOI: Not available
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