The work described in this thesis examines the phosphoinositide (PI) signalling system and its disruption by the anti-manic agent lithium. The effects of lithium upon the accumulation of labelled and unlabelled inositol (poly) phosphates in muscarinic cholinoceptor-stimulated rat brain slices and Chinese hamster ovary (CHO) cells expressing the human M1-muscarinic receptor subtype (CHO-Ml) were examined. Similarly, the effects of this ion on other intermediates of this second messenger signalling system were examined in order to give an overall picture of the action of lithium. These included the accumulation of CMP-phosphatidic acid (CMP-PA), a precursor of the (poly) phosphoinositide lipids, and the agonist-stimulated levels of the (poly) phosphoinositide lipids. Initial experiments examined phosphoinositide metabolism in cortex, hippocampus and striatum to determine whether there were regional variations in both this signalling system and the effects which lithium had upon it. Both cortical and hippocampal PI metabolism were similar, however, striatum was significantly different such that in the continued presence of agonist this region was unable to maintain the initial elevated levels of Ins(l,3,4,S)P4, unlike the other regions examined. Lithium appeared to have a similar disruptive effect on PI metabolism in all regions, with statistically similar EC50 values for the accumulation of InsP1 in all regions. The effects of lithium upon PI metabolism stimulated by a variety of different agonists was examined to determine whether the action of lithium was agonist dependent Lithium appeared to have a similar disruptive effect upon PI metabolism stimulated by these various agonists. Studies examining the effects of lithium upon the carbachol-stimulated accumulation of the inositol (poly) phosphate isomers revealed the presence of a lithium-sensitive accumulation of the inositol bisphosphate Ins(4,5)P2. This study was unable to determine the source of this isomer, however, the formation of this isomer in both cerebral cortex slices and CHO cells suggests the possibility that Ins(4,5)P2 may be a dephosphorylation product of Ins(l,4,5)P3. The lithium-sensitivity of the accumulation of this isomer also suggests that a novel Uthium- sensitive 4- or 5-phosphatase activity may be present in these preparations. The effects of lithium on phosphoinositide metabolism in CHO cells expressing the human M1-muscarinic receptor subtype was also examined to determine whether this cell line would represent a suitable model of cerebral PI metabolism. It was hoped that the use of this cell-line would result in clearer more interpretable data. Indeed, a definitive analysis of PI metabolism in this cell line clearly demonstrated that the addition of lithium to agonist-stimulated cells resulted in a decrease in the intracellular myo-inositol reserves within the cell which resulted in a decrease in the (poly)phosphoinositide precursor of Ins(l,4,5)P3, PtdIns(4,5)P2. In turn this resulted in a time-dependent decrease in the agonist-stimulated levels of Ins(l,4,5)P3 after a lag period of 5-10 min, similar to that observed in cerebral cortex slices. The data demonstrate that the CHO-Ml cell-line is a valuable tool in elucidating the actions of lithium upon PI signalling. In conclusion, the results described in this thesis clearly demonstrate the profound effects that this monovalent ion has on phosphoinositide signalling in the preparations examined. The main action of lithium appears to be the inhibition of the inositol monophosphatase, however, this agent may also have other more subtle effects upon this complex system. These possibilities and their implications are also discussed.

Griseofulvin induces protoporphyria in the mouse. The porphyria is caused by inhibition of ferrochelatase, the last enzyme of the liver haem biosynthetic pathway. Two N-alkylprotoporphyrins can be isolated from liver of griseofulvin-fed mice. One, N-methylprotoporphyrin, is a known ferrochelatase inhibitor. In this thesis NMR spectroscopy was used to determine the structure of the second adduct, N-GfPP, which is an N-monosubstituted protoporphyrin in which griseofulvin (minus a hydrogen atom) is attached to the NC pyrrole of protoporphyrin via an -N-CH2-O- linking group derived from either the 4- or 6-OCH3 group of the drug. A preliminary investigation into possible break-down products of N-GfPP is also described and suggestions made for the mechanism(s) of adduct formation. Individual regioisomers of N-alkylprotoporphyrins with varying N-alkyl groups were synthesised and separated. 1H and 13C NMR techniques were then used to identify structural differences between planar porphyrins and the synthetic N-alkylporphyrins. A discussion has been made of the effect of porphyrin ring currents on proton and carbon resonances. The purification of rat liver ferrochelatase is also described; however only small amounts of highly labile protein were obtained. Experiments devised to optimise the purification procedure also met with little success. Therefore, inhibition studies used cholate-solubilised ferrochelatase and synthetic N-alkylporphyrins. These studies confirm that the ferrochelatase inhibitory activity of an N-alkylporphyrin is dependent on the size and position of the N-alkyl substituent. The 1H NMR study of synthetic N-alkylporphyrins has also been linked to the inhibition analysis. This shows that the majority of N-alkylporphyrin enzyme inhibitors studied have equivalent 6 and 7 propionate groups, whereas the majority of N-alkylporphyrin non-inhibitors have distinctly non-equivalent 6 and 7 propionate groups. A proportion of the N-alkylporphyrins studied do not fit either description, but are still capable of inhibiting ferrochelatase. Mechanisms for the interaction of ferrochelatase with these compounds are therefore described.

It is wall known that Immunoglobulin G (IgG) is transported across the human placenta during gestation. Howewer the route of this transport is not clear and the aim of this thesis is to elucidate the transport pathway. A number of different methods and placental systems were employed to investigate the IgG transport pathway. 1. Human placentae were perfused with a human IgG probe using an isolated cotyledon method, 2. Dissected chorionic villi from both term and first trimester placentae were incubated in culture medium containing a human IgG probe. 3. Human placental cells were cultured and incubated with a human IgG probe. 4. Choriocarcinoma cells (BeUo) were cultured and incubated with a human IgG probe. The IgG transport process was investigated at the light microscope level using tritiated IgG, and at the electron microscope level using colloidal gold particles coated with IgG. From these investigations new evidence was discovered concerning the transport of IgG across the human placenta. In addition an extensive morphological study of the BeWo cells has provided much new information. There are clear indications in the structure of these cells, first of transformed state and secondly of their origin from trophoblast tissue. Observations are presented of the in vitro differentiation of cultured cells towards the villous state of the parent tissue. Cytoplasmic characteristics and mutual positioning of these cells was noted which were similar to those of cytotrophoblast and syncytiotrophoblast in vivo. Bulbous projections were also noted in the culture cells both attached to the tissue and floating in the culture medium. These features may be related to the budding off and depbrtation of trophoblast in vivo.

The multicatalytic proteinase complex (MCP) or proteasome is a high molecular mass (650 kDa) proteinase found in all eukaryotes and a similar particle has been purified from the archaebacteria Thermoplasma acidophilum. MCP can cleave peptide bonds on the carboxyl side of basic, hydrophobic, or acidic amino acid residues, and these activities have been referred to as "trypsin-like", "chymotrypsin- like", and "peptidylglutamyl-peptide hydrolase (Glu-X)" activities, respectively. In this study, the Glu-X activity, assayed with the substrate Z-Leu- Leu-Glu-naphthylamide was investigated. Kinetic studies have shown that it is composed of two distinct components: (a) a noncooperative component (LLE1) obeying Michaelis-Menten kinetics, and (b) a cooperative component (LLE2) exhibiting sigmoidal behaviour. The LLE2 component can easily be distinguished from that of the LLE1 component by the effect of inhibitors, divalent metal ions, KC1, and heat treatment. The addition of 1 mM MnC12 stimulates both components and permits saturation of MCP with substrate at concentrations below the solubility limits of the substrate, under these conditions, a Hill coefficient of 5.2 was calculated for the LLE2 component. Using serine protease inhibitors, peptides as model substrates, and other effectors as probes for the different activities, it was found that (a) the proteinase is in fact a novel type of serine protease, (b) there seems to be at least seven distinct proteolytic activities associated with the complex, and (c) the specificity of the various activities is more complex than first proposed, where the "trypsin-like" activity can cleave after tyrosine residues. Possibility of conformational changes associated with the activation by MnCl2, and with the action of different effectors of the Glu-X were investigated by sedimentation velocity analysis, dynamic light scattering measurements, and electron microscopy of negatively stained preparations. The results provide a direct evidence for conformational changes associated with the activation by MnCl2. Electron microscopic observations indicate a transition from a compact to an extended conformation of the complex in the presence of MnCl2. The hydrodynamic results also provide a structural evidence for the observed positive cooperativity exhibited by the LLE2 component. A correlation between activity and shape of the conformational state of MCP was obtained.

Bovine pancreatic phospholipase A2 (bPLA2) is a 14 kDa (123 amino acids) enzyme with seven disulphide bonds. Its primary function in vivo is the hydrolysis of dietary phospholipids. A T7 RNA polymerase based expression system was used to overexpress a synthetic gene encoding bovine pancreatic pro-phospholipase A2 in E. coli. The expressed protein was directed to the periplasmic space via an outer membrane secretory signal (OmpT) where, after translocation the protein formed insoluble inclusion bodies. Translocation efficiency was significantly increased (from about 25% to over 90%) when protein expression was induced after the cells had reached the stationary phase of growth (O.D.600~2.8). Optimal conditions for refolding of bPLA2 were found to be by rapid dilution under anaerobic conditions, in the presence of 2 mM oxidised glutathione, 4 mM reduced glutathione, 5 mM potassium EDTA and 25 mM sodium borate pH 8.7, with a final protein concentration of approximately 45 mg/L. The final amount of active recombinant protein produced was 22 mg per litre of bacterial culture. Site directed mutagenesis was used to investigate the role of the 58-71 surface loop of bovine pancreatic PLA2 in interfacial binding to a variety of aggregated phospholipids and surfactants. The surface loop was mutated so that the amino acid sequence was similar to that found in porcine pancreatic PLA2. Three mutants were made,Val-63Phe-63 (V63F), Asn-71Glu-71 (N71E) and finally the double mutant (V63FN71E). The mutants were overexpressed in E. coli and refolded in vitro. V63F had an increased affinity for lipid-water interfaces so that its binding to zwitterionic micelles was more like that of the porcine enzyme but catalysis was still similar to the bovine enzyme. Both N71E and V63FN71E showed a reduction in binding to zwitterionic micellar interfaces at pH 8 compared with bPLA2. The affinity for the lipid-water interface could be restored by the addition of a negative charge (via an anionic detergent) to the micellar interface.

1. Mutants of E. coli K12 devoid of acetate kinase and phosphotrans-acetylase activities have been isolated by selection for resistance to fluoroacetate. The properties of the mutants have been studied and the role of the acetate kinase/phosphotransacetylase system clarified. It has a role in acetate excretion and its control properties are consistent with a role in fermentative metabolism. It also has a role in acetate activation at high concentrations of acetate. 2. Studies of acetate incorporation by acetate kinase and phospho-transacetylase-negative mutants of E. coli K12 have revealed the existence of a second system capable of acetate activation in this organism. This system hats properties consistent with a role in the activation of acetate at low concentrations. It is saturated at 2 mM acetate and is also subject to repression by glucose. An acetate thiokinase activity could be detected in cell-free extracts and a number of its properties could be correlated with in vivo observations on the second system. The activity was induced by acetate and repressed by glucose. It had a low Km for acetate. The thiokinase also shows propionate thiokinase activity. Attempts to isolate mutants modified in this activity proved unsuccessful. 3. Problems relating to the study of monocarboxylic acid permeation have been reviewed. The uptake of acetate and lactate by washed cell suspensions of E. coli K12 has been investigated. The data obtained have been considered in the light of criteria required to establish the existence of specific transport systems. Two acetate uptake processes have been found. One shows saturation kinetics with a Km of approx. 10-5 M and its activity is repressed by glucose. The other operating at high acetate concentrations is apparently non-saturable. The effects of a number of factors on acetate uptake were investigated. Preliminary evidence that, under certain conditions, a membrane transport process may be rate-limiting in acetate uptake was obtained. The existence of specific transport systems for D- and L-lactate was established. The properties of these systems were investigated. Lactate uptake was found to be induced by lactate and repressed by glucose. Lactate was not actively concentrated within the cell, but uncoupling agents prevented its transport. The properties of a mutant defective in catabolite repression of L-lactate utilization are described. 4. Mutants of A. aerogenes 1033 devoid of acetate kinase and phospho-transacetylase activities have been isolated by selection for resistance to fluoroacetate. Their properties have been compared with those of the E. coli mutants. The acetate kinase/phosphotransacetylase system has an amphibolic role in A. aerogenes. No evidence for the operation of an alternative mechanism of acetate activation was obtained. The coarse control properties of the acetate kinase/phosphotransacetylase system were studied under a variety of conditions. The role of acetic acid as the inducer of the enzymes of the butanediol-forming system of A. aerogenes was established. Possible relationships between the operation of the acetate excreting system and the operation of the butanediol-forming system are discussed.

The characteristics of the respiratory system of the aerobic, nitrogen-fixing bacterium A.vinelandii were very similar during growth on a medium containing combined nitrogen (urea) compared with growth on nitrogen-free medium (nitrogen-fixing conditions; see Yates and Jones,1974). The growth yield of the organism with respect to ATP (YATP, g cells. mol ATP equivalents-1) was much higher during growth on combined nitrogen medium than during growth on nitrogen-free medium, probably due to the energy requirements of nitrogen fixation. In spite of its outstanding adaptations for growth in aerobic, nitrogen-deficient environments, A.vinelandii shows a poor ability to modify its respiratory system for efficient growth on a source of combined nitrogen. 2. Measurements of ? H+/O quotients (Mitchell, 1966) on batch- grown B.megaterium strains D440 and M suggested the presence of two functional proton-translocating loops in the respiratory chains of these organisms. Substrate-loading experiments (Lawford and Haddock, 1973) demonstrated the presence of proton-translocating loops 1&2 in strain D440 and 2&3 in strain M. The involvement of loop 3 in strain D440 was probably precluded by the absence of cytochrome c from the respiratory chain of this organism. Measurements of the in situ respiratory activity of chemostat cultures of B.megaterium allowed calculation of YATP and M (maintenance coefficient, mol ATP equivalents. g cells-1. h-1) during growth under various culture conditions. YATP values of 13.1 and 10.4 g cells. mol ATP equivalents-1 were obtained for strains D440 and M respectively during glycerol-limited growth. Similar values were obtained during NH4Cl-limited and oxygen-limited growth. These values are considerably lower than the theoretical maximum value for YATP (Forrest and Walker, 1971). Possible reasons for this are discussed.

The isocitrate lyase from a thermophilic Bacillus is activated about threefold by a variety of salts. Such strong stimulation of activity is not seen with isooitrate lyase from the mesophiles, Bacillus licheniformis, Bacillus megaterium, Escherichia coli, and Aspergillus nidulans. The salt activation is markedly pH-dependent. At pH values above 8.6, salt (KC1) indeed inhibits the enzyme activity. Potassium chloride also causes a significant shift of the pH optimum of the enzyme towards the acid side. As the temperature of the enzyme reaction is raised, activation becomes progressively weaker. Potassium chloride also affords considerable protection against enzyme denaturation at 55 C. The activation and the stabilisation, however, appear to be independent effects. Of six other enzymes in the thermophile that were examined, isocitrate dehydrogenase was equally strongly activated by KC1 and malate synthase was less strongly, but significantly activated; citrate synthase, malate dehydrogenase, glutamate dehydrogenase, lactate dehydrogenase were unaffected or slightly inhibited by KC1. The property of being strongly activated by salt appears to be a peculiar characteristic of the thermophile isocitrate lyase and possibly evolved concomitantly with its thermostability. As well as differences in the properties of the thermophile isocitrate lyase and the enzyme from mesophiles, there appears to be a difference in the way in which the enzyme is controlled in the thermophile and the rnesophile, E. coli. The synthesis of isocitrate lyase in the thermophile seems to be induced by isocitrate. A mutant, NG-15, of the thermophile is deficient in isocitrate dehydrogenase, and concomitantly there is a large pool of isocitrate in cells of this mutant. This mutant is able to synthesise isocitrate lyase under most growth conditions, due to the high level of isocitrate in its cells. Further evidence is presented to support this conclusion. There is no evidence in the literature to suggest that the enzyme from E. coli is controlled in this manner. The intermediates of the tricarboxylic acid cycle appear to play an important role in the fine control of the isocitrate lyase from the thermophile, but there is evidence that they are not as important for the fine control of the enzyme from the mesophile, E. coli. Differences have been shown to exist in the properties and mode of control of an enzyme from thermophilic and from mesophilic sources.

A strain of Bacillus stearothermophilus grows on D-gluconate as sole carbon and energy source with a minimum doubling time of 90 min at 55°C. Gluconate is taken up by an inducible transport system, phosphorylated by an inducible gluconokinase and the 6-phosphogluconate formed is metabolised via a constitutive 6-phosphogluconate dehydrogenase. The inclusion of glucose or acetate in growth media represses the formation of activities induced by gluconate, and each is shown to reduce the activity of a preformed gluconate transport system. The isolation and properties of a mutant are described in which gluconate catabolism has lost sensitivity to glucose despite the occurrence of normal glucose catabolism, but which retains sensitivity to the effects of acetate. A mutant devoid of gluconokinase activity was isolated which, after growth in the presence of gluconate, retains high intracellular concentrations of gluconate even after repeated centrifugation and washing. Use of this mutant and of another strain derived from it which grows on gluconate only at elevated temperatures, has allowed study of the gluconate transport system without further gluconate metabolism. The transport system is highly specific, shows a Km of 8 ?M for gluconate and requires energy derived from respiration to perform concentrative transport. Evidence is presented that energy coupling is mediated by a proton gradient. Temporary accumulation of [14C] gluconate occurs under anaerobic conditions in the presence of high intracellular [12C] gluconate concentrations. Evidence is presented that gluconate transport is the rate limiting step for gluconate catabolism during growth and a close correlation between growth rate and the specific activity of the gluconate transport system at different temperatures is reported. It appears possible that a change in the membrane lipid phase may determine the minimum growth temperature of the organism. The gluconokinase has been purified over 600-fold; its properties include high specificity for phosphate acceptor, great stability at 70°C in the presence of magnesium, inhibition by sulphydryl reagents, and a random bi bi kinetic mechanism.

Cell signalling by Receptor Tyrosine Kinases (RTKs) is widely known to regulate several cellular processes. Activated RTKs can associate with many cellular transducer proteins containing PTB (phosphotyrosine-binding) domains and/or SH2 (Src homology 2) domains. The Shc (Src Homology and Collagen) family of adaptor proteins contain both of these critical domains and bind to many growth factor receptors. Four Shc family members have been identified including Shc/ShcA, Sli/ShcB, Rai/ShcC, and the latest member, RaLP/ShcD. Although all the family members share a similar domain modularity, their tissue expression and biological roles are different. As the most recently identified Shc protein, RaLP/ShcD remains poorly characterised. RaLP/ShcD has been shown to have a migratory role in melanoma cells in humans, to interact with Muscle-Specific Kinase (MuSK) receptor in mice, and to regulate cell differentiation during stem cell development. In order to further understand its role in these or other processes, this study aimed to identify novel interacting partners of RaLP/ShcD. The SH2 domain of RaLP/ShcD was shown to interact with another signalling scaffold protein, Gab1, through phosphorylated tyrosine 183, as revealed by GST pull-down and co-immunoprecipitation experiments. Notably, a Gab1Y183F mutant was unable to recruit RaLP/ShcD to the cell membrane in ruffles upon growth factor stimulation. By screening a yeast two-hybrid library,Peg3/Pw1 and HP1α were isolated as novel binding partners for the collagen homology domain 1 (CH1 domain) of RaLP/ShcD. The ability of RaLP/ShcD to interact with both Peg3/Pw1 and HP1α was confirmed by GST pull-down and co-immunoprecipitation assays using transfected human cell lines. Interestingly, a small portion of RaLP/ShcD co-localised with Peg3/Pw1 in the nucleus. Finally, using an affinity column comprising purified CH1 domain coupled to sepharose beads, vimentin was purified from melanoma cell extracts.