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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.

The overall aim of this thesis was to establish relationships between the ion content of isolated rat hepatocytes and metabolic processes such as glucose synthesis and fructose metabolism. In addition, a closely related subject, that of the mechanism of maintenance of the alleged ammonia gradient in liver, was also investigated. Isolated rat hepatocytes were separated from the incubation medium for estimation of intracellular ion and metabolite values. Cell volume was estimated both from wet weight, dry weight and inulin space of cells, and from distribution of tritiated water between cells and medium. Cell volume increased in conditions where intracellular accumulation of amino acids occurred and when cells were treated anaerobically. The specific inhibitor of the Na-K ATPase, ouabain, did not affect cell volume. Hepatocyte K+ and Na+ content, and K+ turnover, were estimated in conditions of added substrates such as fructose, lactate, NH4C1, ethanol, asparagine, glutamine and alanine. Change in turnover of cell K+, estimated with 42K+, was not directly related to change in rate of metabolic processes such as glucose or urea synthesis. However, in all conditions in which increased cell volume occurred and in which amino acids were accumulated, K+ turnover was increased. The effect of added alanine on cell ion and alanine content was examined in detail with special reference to the effects of ouabain and insulin. Metabolism of added fructose led to redistribution of cell and medium inorganic phosphate. Movement of phosphate was concomitant with change in intracellular K+ and Na+ content. Intracellular [ammonia] in isolated hepatocytes incubated without additions was 60 times greater than that of the medium. This constitutes an ammonia gradient only if intracellular ammonia was free in solution and not bound. Mechanisms which might be involved in maintaining high intracellular [ammonial] were investigated.

A comparative survey of the maximum catalytic activities in vitro of 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase in muscles throughout the animal kingdom was carried out. Knowledge of the quantitative nature of the TCA cycle enabled the physiological function and metabolic characteristics of these muscles to be discussed in detail (Chapters Four and Five).

The study of biochemical aspects of brain metabolism is very complex because brain tissue is composed of different cell types. The two major cell types are neurones and oligodendroglia. These two cell types have differing roles but are very closely associated with normal brain development and function. Oligodendroglia are responsible for the synthesis and maintenance of the myelin membrane. In the developing brain, the myelin membrane is formed during a very short period of time. The aims of this study are (i) to investigate age-dependent changes in the activity of the lipogenic enzymes, fatty acid synthetase and palmitoyl-CoA synthetase which are intimately involved in the synthesis and activation of fatty acids for the formation of phospholipids for incorporation into the myelin membrane;(ii) to determine the sub-cellular location of these enzymes;(iii) to investigate the effect of thyroid hormone on the activity of these enzymes and (iv) to determine the route of fatty acid incorporation into myelin lipids. The results presented indicate (i) that the activities of fatty acid synthetase and palmitoyl-CoA synthetase increase during the period of myelination; (ii) fatty acid synthetase is found predoninantly in the cytoplasmic fraction from brain homogenates, whereas palmitoyi-CoA synthetase activity is present in all menribrane fractions, although activity measured in myelin may be due to contamination; (iii) the administration of thyroid hormone has no effect on palmitoyl-CoA, synthetase, whereas its specific antagonist, propylthiouracil, causes a decrease in enzyme activity in the mitochondria enriched fraction; (iv) the accumulation of radioactivity in the myelin fraction from labelled fatty.: acid involves all subcellular organelles including the cytoplasm and no precursor-product relationship is apparent. The results will be considered in the light of data available for lipo- genic and other enzymes involved in lipid synthesis.