Proteins of the sympathetic nervous system

This thesis describes studies of the membrane proteins of bovine adrenal chromaffin granules (the catecholamine-storing particles of chromaffin cells), of the proteins of the related "large dense-cored vesicles" of bovine splenic nerve axons and of protein synthesis in rat superior cervical ganglia.
1. Chromaffin Granule Proteins.
Electrophoretic Experiments. After initial experience with the electrophoretic system described by previous investigators for the analysis of chromaffin granule membrane proteins (which utilises 1% SBS in borate-buffered 3.5% polyacrylamide gels, pH 9.6) an improved electrophoretic system was developed and used for molecular weight studies. The improved method involved electrophoresis in 6% gels containing 0.1% SDS and 0.1M sodium phosphate buffer pH 6.5, followed by fixation in 25 trichloroacetic acid, before staining.
This method was used to determine the electrophoretic behaviour of unreduced chromaffin granule membrane proteins, the majority of which were found to have anomalous mobilities which did not follow the predictions of the Ferguson equation. The anomalous proteins were shown, by staining by the periodic acid/Schiff reagent method, to contain carbohydrate. One protein which contained no carbohydrate and which exhibited regular electrophoretic properties was chromomembrin B, one of the two major membrane proteins. This protein was estimated to have a molecular weight of 28,500 from measurements in 6% polyacrylamide gels. It was unaffected by treatment with dithiothreitol.
Quantitative densitometry was used to try to determine the degree of contamination of the membrane preparation with residual soluble proteins. The major soluble protein, chromogranin A, was isolated in a highly-purified form by gel-filtration and ion-exchange chromatography and used to calibrate the densitometer. It was found that ca. 13% of the protein in an SDS-extract of chromaffin granule membranes was chromogranin A and this result was discussed in the light of various conflicting literature claims.
Ion-exchange chromatography was also used to prepare a homogeneous sample of soluble dopamine β-hydroxylase. This sample was reduced and run in the phosphate/SDS gel system, when it was found to give only one stained band. It was suggested that earlier reports that this enzyme gives two sub-units on reduction were erroneous and that the "second" "sub-unit" was probably a contaminating protein. This experiment gave a molecular weight of ca. 73,500 for fully-reduced dopamine β-hydroxylase end it was suggested that the native enzyme (which has a molecular weight of ca. 290,000) was probably tetrameric. The electrophoretic behaviour of the SDS-denatured, unreduced fora of the enzyme was discussed on this basis and it was suggested that this fora might show considerable residual molecular asymmetry, resulting in a false value for the molecular weight if the determination was carried out in gels in the absence of reducing agents.
Gel-filtration Experiments. With the improved resolution afforded by the phosphate/SES electrophoretic system, it was found that the Sephadex G-200 column method described for the purification of the two major membrane proteins did not yield completely homogeneous samples, and a series of experiments were carried out to establish the optimum bed material for gel-filtration separation of the proteins. The best separation was achieved with Sepharose 6B resin in a 90cm × 8cm2 column and this system was used preparatively to make milligramme quantities of chromomembrin A and chromomembrin B. Even with this improved gel-filtration procedure, however, a completely pure preparation could not be obtained in one chromatographic step and the partly-purified proteins were each chromatographed for a second time on the sane column, after ultrafiltration to a suitable volume. Electrophoretic analysis showed that, even after this step, none of the proteins had been isolated in a pure state, and it was concluded that earlier claims in the literature that this end had been achieved with a single separation on Sephadex G-200 were open to doubt. Additionally, it was found that one of the characteristic proteins of the granule membrane – the cytochrome – was degraded during this prolonged isolation process, with the loss of its identifying spectral properties.
Molecular weight values obtained by calibrating the large column with unreduced standard proteins were found to agree rather well with those obtained by electrophoresis, unreduced chromomembrin A once again shoving an apparent molecular weight of ca. 135,500, which is not obviously related either to the molecular weight of the native enzyme or to that of the fully-reduced sub-unit.
2. Splenic Nerve Vesicle Proteins
Samples enriched with large dense-cored vesicles, prepared from bovine splenic nerve axons in the Karolinska Institute and transported to Oxford, were osmotically shocked with tris-sodium succinate buffer (0.005M, pH 5.9) and the soluble proteins were examined, using the tris-glycine electrophoretic buffer system. The soluble proteins from the fractions thought to be enriched with nerve vesicles showed a stained band with the mobility of dopamine β-hydroxylase, one of the proteins believed to be located in these vesicles, but only a very faint band was observed in the position expected of chromogranin A. This observation was confirmed using repurified fractions and in these fractions it was particularly noticeable that the dopamine β-hydroxylase band appeared to be stronger in the leas dense of the two gradient-fractions (RII). This observation was related to the possibility of vesicle damage during the isolation process. The faintness of the chromogranin A band in these samples was at first puzzling, but this was explained by an earlier report in which it was found that splenic nerve vesicles contained about 60 times as much dopamine β-hydroxylase, when expressed in terms of the noradrenaline content, as adrenal medullary chromaffin granules (whose soluble proteins show a prominent chromogranin A band).
When the insoluble proteins of the splenic nerve vesicle fractions were solubilised with SDS and analysed, a much more complex pattern was observed than is found for the membrane proteins of chromaffin granules. Two possible explanations could be envisaged for this observation: either the granule membrane of the splenic nerve vesicle is much more complex, in terms of protein composition, than that of the chromaffin granule, or the preparations were quite heavily contaminated by proteins of non-vesicular origin. The latter explanation may be indicated by Lagercrantz's own biochemical studies, which indicated a purity of ca. 18%, and this is supported by the fact that one of the most prominent bands in the electrophoretic patterns of both soluble and insoluble proteins had a mobility close to that expected for bovine serum albumin.
3. Protein Synthesis in Rat Superior Cervical Ganglion.
Use was made of the separative capacity of the phosphate/SDS/6% polyacrylamide gel electrophoretic system to study newly-synthesised proteins in rat superior cervical ganglia incubated with radioactive leucine. After electrophoretic separation of the proteins, gels ware chopped into 1.0mm slices with a sectioning-apparatus and extracted with Soluene 100 tissue solubiliser, to release the protein-bound radioactivity, which was estimated by scintillation-counting. Since published details of the overall reliability of the method were meagre, some emphasis was placed on the evaluation of the possible sources of error in each of the stages in the analysis leading to the construction of the "labelling-profile". The pipetting, electrophoresis and scintillation-counting steps were found not to introduce significant errors into the profiles, or to distort recoveries appreciably, end the least reliable stage in the analysis was found to be the mechanical sectioning of the frozen gel, which may give rise to both random and systematic errors. While the random errors arising from variations in section-thickness could be estimated as being about 5%, the systematic error was inherent in the method of sectioning and could not be determined. This error arises from the fact that the gel cannot be accurately aligned with the razor-blade at the commencement of sectioning and from more subtle effects which follow from trying to represent a continuous distribution by a discontinuous one. A computer-simulation model of the sectioning process was developed which indicated the way in which these more subtle errors might distort the labelling-pattern under those circumstances in which the slice-thickness approaches the inter-peak spacing and the peak width. Although reducing the section thickness would be expected to reduce this kind of sectioning error, it was shown experimentally that a redaction in the slice thickness from 1.0 to 0.6mm did not have much effect on the observed resolution, whereas it lowered the accuracy with which the dpm in each slice could be determined and much increased the labour of the experiment.
The other main source of variation between experiments lay in the efficiency of the Soluene-extraction process used to release the protein-bound radioactivity into the bulk phase, where it could be counted. Different batches of Soluene were found to give widely-differing recoveries of radioactivity and, since it was necessary to elute the radioactive label from the gel matrix before it could be counted even when using 14C-leucine, it would seem likely that the variation in the recoveries was caused by variable extraction-efficiency. It was, however, found that the reduced extraction did not, in general, lead to some proteins being extracted preferentially but, instead, caused a general depression of the whole profile.
A number of experiments with both 14C- and 3H-leucine served to show that the incorporation of label by normal resting ganglia gave rise to a characteristic double-jumped labelling-profile which was more or less evident according to the quantity of radioactivity applied to the gel, high loadings giving patterns which were rather exaggerated although still showing peaks at the same relative mobility values. A reduction in the labelling-period fros 2.5 hours to 20 min did not lead to much increase in the selectivity of labelling, as judged from the observed pattern, but necessitated the use of 3H-leucine of high specific activity, with associated problems in the scintillation-counting. Experiments involving stimulation of the ganglia, possibly together with chronic denervation, failed to reveal any marked alteration in the labelling-pattern in response to changes in the environment of the ganglia and this lack of effect was discussed in the light of possible changes in the size of the intraganglionic pool of leucine and its specific activity. In spite of the relative lack of apparent control of the rate of synthesis by environmental factors, it was thought likely that the labelling-profile does in fact represent newly-synthesised secretory (soluble) proteins.

Advisor: Smith, A