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The amino-acid composition of gelatin has been extensively studied, but until recently little attention has been paid to that of its precursor collagen, the natural protein of skin. Some of the analyses of collagen which have been reported were carried out by methods which are now considered unsatisfactory, and in no case have most of the major component amino-acids been determined in one sample of the protein. Further, most of the analyses have been made on coUagens which had been given an alkaline or enzyme treatment, or both, to remove the keratins, elastic fibres and reticular tissue which occur in close association with it, and there is a risk that such treatments will cause some modification of the collagen. For the present study, a sample of collagen has been prepared with the minimum of chemical treatment, since it was considered that the presence of small amounts of elastic fibres and reticular tissue would lead to less error than the treatments necessary to bring about their removal. Also, it seems doubtful whether it is possible to remove these proteins completely by any of the methods which have been suggested.

The greater part of this work describes the biochemical and morphological changes accompanying grain development in cereals. Attention is focussed on those aspects most likely to be involved in the regulation of grain yield and composition. Thus, since starch is the major component of grain yield much of the work is concerned with the developmental enzymology of carbohydrate synthesis. The composition, properties and structure of the developing starch granules and amyloplasts have also been described. Grain development is associated with a range of catabolic processes and a number of enzymes active in carbohydrate degradation has been identified. Although oxygen levels in the developing endosperm are unknown, recent work suggests that the mitochondria present are fully func- tional. It is possible that oxygen required for grain respiration is derived from pericarp photosynthesis. At the same time this tissue may fix carbon dioxide derived from endosperm respiratory processes. Thus, the pericarp may be involved in the overall control of endosperm metabolic activity. Studies of endosperm cell division indicate that this continues for more than half the developmental period, later divisions producing mainly aleurone cells. Hence, stimulation of endosperm cell division early in development will increase the yield of carbohydrate whereas at later stages it will increase the yield of protein. Further studies describe the pattern of mineral ion accumulation, the growth and nutrition of the develop - ing embryo and the morphological changes in the crease region through which nutrients move from the vascular tissue of the pericarp to the endosperm. A second group of papers is concerned with the control of a- amylase and polyphenol oxidase activity during cereal grain germination. Finally, there is a small group of papers describing research on the properties of photosynthesising tissues including marine algae.

Mutual Information (MI) based methods are used to predict contact residues within proteins and between interacting proteins. There have been many high impact papers citing the successful use of MI for determining contact residues in a particular protein of interest, or in certain types of proteins, such as homotrimers. In this dissertation we have carried out a systematic study to assess if this popularly employed contact prediction tool is useful on a global scale. After testing original MI and leading MI based methods on large, cross-species datasets we found that in general the performance of these methods for predicting contact residues both within (intra-protein) and between proteins (inter-protein) is weak. We observe that all MI variants have a bias towards surface residues, and therefore predict surface residues instead of contact residues. This finding is in contrast to the relatively good performance of i-Patch (Hamer et al. [2010]), a statistical scoring tool for inter-protein contact prediction. i-Patch uses as input surface residues only, groups amino acids by physiochemical properties, and assumes the existence of patches of contact residues on interacting proteins. We examine whether using these ideas would improve the performance of MI. Since inter-protein contact residues are only on the surface of each protein, to disentangle surface from contact prediction we filtered out the confounding buried residues. We observed that considering surface residues only does indeed improve the interprotein contact prediction ability of all tested MI methods. We examined a specific "successful" case study in the literature and demonstrated that here, even when considering surface residues only, the most accurate MI based inter-protein contact predictor,MIc, performs no better than random. We have developed two novel MI variants; the first groups amino acids by their physiochemical properties, and the second considers patches of residues on the interacting proteins. In our analyses these new variants highlight the delicate trade-off between signal and noise that must be achieved when using MI for inter-protein contact prediction. The input for all tested MI methods is a multiple sequence alignment of homologous proteins. In a further attempt to understand why the MI methods perform poorly, we have investigated the influence of gaps in the alignment on intra-protein contact prediction. Our results suggest that depending on the evaluation criteria and the alignment construction algorithm employed, a gap cutoff of around 10% would maximise the performance of MI methods, whereas the popularly employed 0% gap cutoff may lead to predictions that are no better than random guesses. Based on the insight we have gained through our analyses, we end this dissertation by identifying a number of ways in which the contact residue prediction ability of MI variants may be improved, including direct coupling analysis.

Recent evidence has suggested that oncogenic transformation of mammalian tissue culture cells results in altered expression of several surface components. One of these changes is the reduced ability of transformed cells to synthesise complex gangliosides. This modification has been correlated with the other phenotypic alterations of transformed cells and with their tumorigenic potential. We have adopted a novel approach to elucidate the relationship between the impaired synthesis of complex gangliosides in transformed cells and other aspects of the transformed phenotype. Mutants of Balb/c 3T3 mouse fibroblasts, which normally contain substantial levels of the gangliosides GM3. GM2. GM1 and GD1a. have been selected for failure to express ganglioside GM1 at the cell surface. Other phenotypic properties of these mutants have been investigated, to determine whether the expression of any of the characteristics normally associated with transformed cells are dependent on the loss of ability to synthesise GM1 or the more complex gangliosides, GD1a and GTla. Cholera toxin, which is known to bind specifically to ganglioside GM1 with high affinity, has been used in the selection of these mutant cell lines. A method involving antibody-dependent complement-mediated lysis of cells able to bind cholera toxin was used as the one of choice. Previously, there has been some doubt as to whether any other cell surface receptors for cholera toxin exist in Balb/c 3T3 fibroblasts. Evidence is presented in this thesis, and in an accompanying paper, that the major receptor for cholera toxin in these cells is a ganglioside with similar characteristics to GM1. We have further demonstrated that a substantial proportion of this lipid is insoluble in neutral detergent and remains with Triton X-100 extracted cytoskeletons of Balb/c 3T3 cells. The possibility that GMlis indirectly associated with the cytoskeleton is discussed.

A particulate enzyme preparation made from 1-week-old dark-grown pea (Pisum sativum) seedlings has been shown to incorporate glucuronic acid from UDP-D-[U-14C]-glucuronic acid into a hemicellulosic product. Optimum conditions for the incorporation include the presence of Mn2+ ions between 4 and 10 mmol dm-3 and a pH of approximately 7.5. UDP-D-xylose at 1 mmol dm-3 allows incorporation to continue for at least 8 h. In its absence, the reaction stops within 30 min. Analysis of the product by partial and total acid hydrolysis, followed by paper chromatography or electrophoresis, indicates that the polysaccharide produced is a glucuronoxylan.

This thesis describes a project which had a simple central objective: to take an existing preparation of dissociated cells from the immature cerebellum and develop conditions in which these cells could survive and differentiate in monolayer culture. The aim was to develop a system in which brain development could be investigated, particularly at the biochemical level in a simplified environment. The study of cellular differentiation in vitro has two advantages: 1. Aspects of a cell's development which are intrinsically programmed in the cell at that stage can be distinguished from aspects which are dependent on some extrinsic influence, for instance from another cell type. 2. Cultures have a more restricted cellular composition than the brain and therefore may go some way to overcoming the cellular heterogeneity of brain tissue which renders interpretation of most conventional biochemical measurements difficult. To realise the first it is necessary to compare a cell's differentiation in vitro with its normal course of development in vivo. To realise the second, cultures must be of a simplified and defined composition in which biochemical measurements can be attributed to particular cell types. Both requirements imply the use of cultures which contain in quantified proportions a few cell types which can be specifically identified by reliable criteria, rather than in terms of such general categories as 'neurons'. The use of a single area of the brain, the cerebellum, was an important first step in restricting the cellular composition of the cultures. The cerebellum contains large numbers of relatively few neuronal types which differ greatly in perikaryal size and therefore were amenable to a size-based separation of cell types by unit gravity sedimentation. Culture conditions were established for the survival and differentiation of the mixed cerebellar cell suspension and some of the separated cell fractions. This led to cultures containing a high proportion of granule neurons. However a fraction enriched in Purkinje cells did not survive well in culture. Finally, the composition of the cultures was further defined in a study involving cell type-specific immunological markers and the different uptake of the inhibitory neurotransmitter, GABA. The description of this work is organised in seven more or less self-contained chapters, and can be informally divided into two parts. Chapter 1 is a general introduction to the problems of studying development, to the advantages of the cerebellum, in particular the detailed knowledge of some mechanisms of cerebellar development gained from work with mutants and X-irradiation and to the current knowledge, or lack of it, concerning the molecular basis of cellular interactions. Chapter 2 is a description of the cell isolation method used here, and of some improvements to the method effected during the course of this work. Chapter 3 completes the first part of the thesis and deals briefly with the history of brain cell separation techniques before detailing the separation of cerebellar cells by a unit gravity sedimentation technique. This was a joint project with James Cohen and Gary Dutton at The Open University and Graham Wilkin and Robert Balazs at MRC, Carshalton. Chapter 3 concentrates mainly on the part of the project which was the primary work of this author, that is the size analysis of cells using a Coulter counter and points out those parts of the work, in particular electron microscopy, which were performed by others. The second part begins with Chapter 4, which is a review of nervous system culture work with the aim of establishing whether there are reasons why the contribution of cell cultures to our biochemical knowledge has been quite limited, although they have been in use for many years and would appear an ideal controlled system for biochemical studies. Chapter 5 is a description of the search for culture conditions in which one week postnatal cerebellar cells would survive and grow, and of their pattern of development in culture. Chapter 6 details the use of markers to define culture composition more closely; cerebellar cultures were found to be over 80% in a single neuronal type, the granule cell. Cultures of embryonic cerebellum were found to contain a distinct population of neurons which are absent from cultures of older animals, Chapter 7 briefly summarises some future directions for this work and suggests that defined cell culture systems in alliance with modern immunological techniques, may offer the best route to find the elusive cell surface recognition molecules which are generally believed to mediate the cellular interactions that control development. Some of this work, together with related aspects which are primarily the work of others, has been published elsewhere. Copies of these papers are attached.

Yeast 3-phosphoglycerate kinase (PGK), a glycolytic enzyme, was isolated, purified and characterised, and subjected to cyanogen bromide cleavage. Three of the four cyanogen bromide fragments were purified by gel filtration, and enzymically digested with trypsin, chymotrypsin, pepsin, thermolysin and staph. protease. The resulting peptides were purified by high voltage electrophoresis on paper, and were sequenced by the dansyl-Edman technique. Of the 243 amino acids constituting these three cyanogen bromide fragments, 221 were placed in order by direct sequencing, composition, and comparison with the horse muscle enzyme sequence, with which yeast PGK exhibits an estimated 65% identity. By fitting the sequence to the 2.5 R-resolution electron density map of yeast PGK, the positions of the reactive alutamyl residue (Glu-403) and of the reactive tyrosyl residue (Tyr-195) have been located, at sites distant from that of bound ATP. The remote location of the glutamyl residue supports the suggestion that PGK must undergo a substrate-induced conformational change in order to bring Glu-403 into closer proximity with the ~-phosphoryl of ATP. The glycine lining of the ATP-binding pocket of the horse muscle enzyme is conserved in the yeast enzyme, as are the glycines near the t-phosphoryl of bound ATP. However, the location of the hinge region of the molecule, tentatively identified in the horse muscle enzyme sequence, is not conserved in the yeast PGK sequence. The structure of PGK has been compared with that of other kinases, and similarities with hexokinase and adenylate kinase noted.

The aim of the study was to determine the efficiency with which the carbon skeletons of essential amino acids are utilised by the pig when they are limiting in its diet. This requires the oxidation rate of an amino acid to be measured accurately using a tracer labelled with an isotope of carbon or hydrogen. The use of carbon labelled tracers requires the animal to be enclosed in a sealed chamber for the collection of CO2; to avoid this the use of [3H]-labelled tracers was investigated. The oxidation rates of [14C]-labelled tracers when the tracee was limiting and the animal's nitrogen intake was 2 g/kg0.75.d, showed that leucine was used with an efficiency of 0.88; for phenylalanine and histidine the efficiency was over 0.99. When the animal's nitrogen intake was reduced to 0.23 gN/kg0.75.d, and the limiting amino acid was removed entirely from the diet, the oxidation rates did not change significantly. The oxidation rates of [3H]amino acids were very variable compared to the [14C]amino acids and the techniques used were critically analysed. Three reasons were identified for the differences between the [14C]- and [3H]tracers. These were the measurement of tritiated water, the purity of the infused tracers and the position of the isotope in the labelled amino acid. The first two were solved by the adaption of techniques to the high accuracy required. The third reason was a problem of isotope becoming incorporated into synthetic pathways via exchange reactions. No amino acids were available labelled with a [3H] atom which would not be incorporated; therefore it was necessary to synthesise one. L-[33H]valine was chosen because a suitable precursor for its synthesis, L-[2,3-3H]valine was available and because L-[1-14C]valine was also available for comparison The L-[2,3-3H]valine was acetylated to produce N-acetyl-D, L-[3-3H]valine which was then hydrolysed by the stereospecific enzyme acylase I to yield N-acetyl-D-[3-3H]valine and L-[3-3H]valine. The products were separated using a cation exchange column and the L-[3-3H]valine was purified by paper chromatography. Contamination with D-[3-3H]valine was measured using D-amino acid oxidase and L-[2,3-3H]valine using L-amino acid oxidase. The oxidation rates of L-[1-14D]valine and L-[3-3H]valine were compared in the rat and the oxidation rate of L-[3-3H]valine was significantly higher. The experiment was repeated using L-[3-3H]valine synthesised by Amersham International plc. using the same method; the structure was established by NMR. In this case both tracers were oxidised at the same rate. The difference in the first experiment was therefore probably due to slight contamination of L-[3-3H]valine with L-[2,3-3H]valine, the tritium in position 2 being removed by reversible transamination of valine, i.e., not only during its oxidation. Finally, the two tracers were compared in the pig. It was found that the valine flux rates calculated from the two tracers were in closer agreement when a correction was made for infused D-labelled valine. Using the techniques developed throughout this work, L-[1-14C]valine and L-[3-3H]valine measured the same rate of valine oxidation. L-3-3H]valine was a more precise tracer of valine oxidation.

In chapter one the literature of androst-16-enes is reviewed, showing how the economic importance of these steroids is due to both porcine physiology and human sensibilities. Their biosynthesis and physiological function in the pig are discussed showing their relationship to meat producting potential. Their effects upon meat and consumer opinion are detailed together with a discussion of the drawbacks of conventional methods of taint elimination. The chemical generation of Δ¹⁶ bonds is also reviewed with a discussion of the limitations of various methods. Lastly the idea of auto-immunization is explained showing how it may have an important role to play in the solution of the boar taint problem. In chapter two a ring C derivative of androstenone, its llα hemisuccinate, was synthesised from adrenosterone in seven steps. The catalytic hydrogenation and selective ketalization procedures used in the first two steps were optimised. Conversion of the 17-ketone to a Δ¹⁶ bond by Barton's reaction of hydrazones requires sodium reduction of a vinyl iodide to yield the olefin and these conditions caused the reduction of the 11-carbonyl to an llα-hydroxyl group. This was esterified with succinic acid, and after hydrolysis of the 3-ketal, gave the desired hemisuccinate. In chapter 3 a ring B substituted androstenone, its 7-carboxymethoxyloxime, was synthesised from DHEA (androst-5-ene-3β-ol-l7-one) in eleven steps. Ring B was functionalized by allylic oxidation of the Δ⁵ bond to an unsaturated ketone. Removal of the Δ⁵ bond by catalytic hydrogenation and protection of the 7-carbonyl allowed the selective conversion of the 17-ketone to a Δ¹⁶ bond. Deprotection of the 7-ketone, its condensation with carboxymethoxylamine, and oxidation of the 3-hydroxyl to a carbonyl group yielded the product. The use of MS and ¹³C NMR data in differentiating between the ketones and ketals produced is demonstrated. In chapter 4 an isomer of androstenone with inverted configuration at C₁₄ (14β) was synthesised from 5α,14α-androstan-3β-ol-17-one using various synthetic strategies. The final route chosen allows for high yield inversion at C₁₄ in five simple steps. Unsaturation is introduced by bromination and dehydrobromination, but the spontaneous isomerisation of the initial product requires conversion of the isomers produced to a common product, the Δ¹⁴ -7 ketal, before catalytic hydrogenation is used to give the inverted configuration Hydrolysis of the ketal allows the conversion of the 17-carbonyl group to a Δ¹⁶ bond by Barton's reaction of hydrazones. The 3β-hydroxyl was oxidised to a carbonyl group with chromic acid to give 5α,14β-androst-l6-ene-3-one. In chapter five an interesting epoxidation observed during work on ring B functionalization is examined further. Under the same conditions as a Δ⁵ bond is allylically oxidised a Δ¹⁶ bond is epoxidised. Studies with simple analogues and analysis of the by-products of earlier oxidations suggest that an explanation more complicated than ring size or simple steric hindrance is required. This is confirmed by the relationships between the differences in reaction products and molecular conformation observed upon inversion of configuration at C₁₄. The results are explained in terms of a recent theory on the mechanism of oxidations by metal oxides. This requires consideration of the relationship between the orientation of the axial allylic proton and the hindrance at the olefinic bond. In chapter six an unexpected reduction led to the proposal that under certain conditions hydroxylamine can condense to form diazene, a known reductant. A recent paper upon a similar system contained a mechanism that would only explain both sets of results if modified substantially. The reasoning behind these modifications is given, along with a possible explanation for other anomalous results in this chapter.

The physiological significance of indole-acetyl aspartic acid (lAAsp) in auxin metabolism was examined with special reference to its role as part of a homeostatic mechanism to control hormone levels in pea seedlings (Pisum sativum L.)• A technique is described for the chemical synthesis of lAAsp. This was successfully used to obtain samples of the authentic compound. Techniques for the isolation, purification and concentration of lAAsp from plant tissues were examined in detail and a technique giving improved recoveries of the compound is described. Several techniques were used in an attempt to obtain more reliable identification of the presumed lAAsp in plant extracts. These included paper and thin layer chromatography in several solvent systems; mass spectroscopy (MS); gas chromatography/mass spectroscopy (GC-MS) and acid and alkali hydrolysis of the isolated compound. Although the presumed lAAsp consistently co-chromatographed with samples of the authentic compound, technical problems during isolation and derivatization prevented its successful identification by MS and GC-MS. When the presumed 14C-labelled lAAsp was reapplied to intact pea plants, 14C was found with indoleacetic acid (lAA) indicating that the conjugate could be hydrolysed at the presumed peptide bond to release the free hormone. The formation of lAAsp by plants may therefore represent a means of storing excess lAA in a protected and inactive form from which it can be released if tissue concentrations fall' The time course of uptake, conjugation and export of 14C-IAA following its application to the apfcgs of intact pea seedlings was examined in relation to photomorphogenesis and dwarfism.

In a model study concerned with the mechanism of glycosidase action, six N-glycosylimidazoles and five N-(tetra- O-acetyl-glycosyl) imidazoles have been synthesized by the condensation of the appropriate tetra-O-acetyl-glycosyl halide with imidazole. These compounds have been characterised by their elemental analysis, optical rotation and NMR spectra. In all cases the beta-anomeric compounds predominated over those with alpha-configuration in the reaction products. Analysis of 1H NMR data was carried out on a first order basis,and also by non-iterative and iterative calculation procedures in order to establish the conformations of the N-glycosylimidazoles. The analysis revealed that under certain conditions, in four compounds the sugar pyranose ring is distorted from the normal 4C1 conformation. This distortion has been interpreted in terms of the operation of the reverse anomeric effect. The hydrolytic stabilities of 1-alpha- and beta-D-glucopyranosylimidazoles have been examined in water, pH 3.0 sodium formate buffer, O.56M formic acid, 6N-hydrochloric acid, 10N-sulphuric acid and 1N NaOH. Optical rotation measurements, paper chromatography and analysis for estimation of glucose failed to detect any evidence of hydrolysis. This result is strong evidence against the nucleophilic intervention of a histidinyl side chain in glycosidase action. Hydrolytic cleavage was also attempted with solutions of (yeast) alpha- and (almond) beta-glycosidases, and no glucose was released under conditions in which standard compounds methyl alpha- D-glucoside and cellobiose were hydrolysed significantly. The ORD/CD curves of N-glycosylimidazoles and a series of p-nitrophenyl-glycosides have been examined as an aid to establishing anomeric configurations for N-glycosylimidazoles. In general anomeric pairs of compounds show ORD/CD curves of opposite sign, but some exceptions have been observed in the case of N-glycosylimidazoles. 1-alpha- and beta-D-glucopyranosylimidazoles were found to possess inhibitory properties towards the enzymes (yeast) alpha-glucosidase, (almond) beta-glucosidase and lysozyme. These properties have been investigated in detail. The alpha-anomer shows an unexpectedly high degree of inhibitory behaviour.