Studies on neuronal proteins

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 (0.3 M 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 × 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 × 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. These results showed that the only intracellular acetylcholinesterase activity which was demonstrable with this technique was within elements of the endoplasmic reticulum (agranular in the axons and granular in the chromaffin cells). Apart from the finding that most chromaffin cells contained appreciable amounts of the enzyme, three other notable findings were made using this technique. First, acetylcholinesterase was never found within nerve terminals. Secondly, there were occasions when the acetylcholinesterase-rich endoplasmic reticulum appeared to be in the process of fusing with either the axonal or chromaffin cell's plasma membrane. On such occasions, the reaction product within the tubules seemed to be continuous with that outside the cell. Finally, there was demonstrable activity outside adjacent cells only when at least one of them contained intracellular activity.
4. Extracellular fluid levels of acetylcholinesterase.
Serum: The levels of the enzyme were first examined in the sera of calves and sheep of widely differing ages. It was found that the level in bovine serum was very high some 20 days before birth (the earliest time studied) and that from this peak there preceded a seemingly exponential decline in the serum level up to about 15 years of age (the oldest animal studied). In between these extremes it was found that the levels of the enzyme varied according to the health of the animal, the gestation time at parturition, and whether the birth was normal or induced. Sheep samples also showed a downward trend in the later stages of gestation, but they differed from the calf in that the acetylcholinesterase level of the newborn lamb was already the same as the concentration in the serum taken from adults. These results are discussed in relation to the development of the nervous system rather than to neonatal physiology.
Cerebrospinal fluid: It was found that the concentration of acetylcholinesterase in rabbit cerebrospinal fluid could be altered by stimulating various peripheral nerves (stimulation of these nerves was known to release acetylcholine from some structures within the brain). An electrophoretic analysis of the cerebrospinal fluid revealed the presence of only one isoenzyme of acetylcholinesterase. It was found that the relationship between the brain and the cerebrospinal fluid was very similar to that which existed between the adrenal and its perfusate; there were several soluble isoenzymes in the brain but only one of these was released into the cerebrospinal fluid.
5. Turnover of acetylcholinesterase.
This was studied from a combined biochemical and cytochemical point of view. The recovery of enzyme activity in the superior cervical ganglion of the rat after poisoning with DFP was found to be complex, consisting of at least two parts. However, in the initial stages, when the recovery was most rapid, all of the enzyme activity appeared to be restricted to the postganglionic cells. Thus, postganglionic adrenergic cells are capable of synthesizing acetylcholinesterase very rapidly. There was no selective reappearance of any of the isoenzymes, nor was there found to be any selective disappearance induced by denervation. Cytochemistry revealed that, in common with splanchnic axons, there was never demonstrable enzyme activity within the nerve terminals of the normal preganglionic fibres. These results are used to discuss the importance of the contribution of preganglionic fibres to the total acetylcholinesterase activity of ganglia.