1. The literature on the egg white proteins has been reviewed. 2. The individual egg white proteins have been isolated and the methods used described. 3. The possible origin of these proteins has been discussed. An attempt has been made to elucidate this problem by refined electrophoretic techniques. No definite conclusion could be reached with the methods available. 4. The N-terminal residues of the individual proteins were examined and the results found to be in good agreement with the literature. 5. The structure of the native ovalbumin molecule has been examined by both chemical and physical means during progressive denaturation in alcohol, urea and guanidine hydrochloride. The results of the two investigations were shown to be in agreement with one another. 6. A 'six-shaped' structure has been proposed for the configuration of the native ovalbumin molecule. 7. This structure involves the hypothesis of an intra-molecular link since there is known to be a C-terminal, but no N-terminal amino acid residue. The disulphide bridge suggested by Anfinsen and Redfield (1956) hg.B been shown to be unlikely. This link has not been identified as a chemical entity although it has been suggested that low pH enables hydrogen bond breakers to rupture the molecule more easily.

1. YADH was immobilised on two aminoethylcelluloses and their properties were compared. 2. Experimental conditions for the coupling of YADH to Cellex-AE were optimised; these conditions were used for immobilising LDH and MDH on Cellex-AE. 3. The effect of sodium borohydride reduction on the stability of YADH, LDH and MDH immobilised on Cellex-AE was studied. 4. The effect of pH on the stability of soluble YADH, LDH and MDH was studied. The results were compared to those for the three enzymes immobilised on Cellex-AE. 5. The effect of temperature on soluble YADH, LDH and MDH was compared to the effect of temperature on several immobilised derivatives of these enzymes. 6. YADH was immobilised on DEAE-cellulose. The pH variation of the kinetic parameters of the immobilised derivatives were compared to those of soluble YADH. 7. LDH was immobilised on both Cellex-AE and PEI. The kinetic parameters and stabilities of these two derivatives were compared. 8. LDH was immobilised on NP/3 Nylon Powder. The pH variation of the kinetic-parameters of the immobilised derivatives were compared to those of soluble LDH. 9. The change in equilibrium constant when YADH and LDH were immobilised on macromolecular supports was studied. A theory was developed to explain these changes.

The chemistry required for the immobilization of enzymes onto the inner surface of nylon tubing is demonstrated and conditions are optimised. Nylon tube immobilised enzymes are then introduced into continuous-flow systems so that their flow characteristics can be determined and the chemistry of immobilization best suited to continuous-flow analysis ascertained. The practical use of immobilized enzyme derivatives is evaluated in four separate methodologies end their stability and pH dependence is assessed.

NF-KB/Rel transcription factors and IKB proteins play a central role in the rapid induction of genes transcribed in response to a variety of extracellular stimuli. Signal induction results in the phosphorylation and ubiquitination of IKBalpha a prior to proteasomal degradation and release of NF-KB. The objective of this work was to identify residues within the N- and C-termini of IKBalpha which were important for the function of the protein, specifically the residues contacting the p65 subunit of NF- KB and those involved in the turnover of IKBalpha. In addition, the structure of the C-terminal region of IKBalpha was also examined to gain a better understanding of the functional properties of this domain. The identification of conserved stretches of acidic residues in the C-terminal region of IKBalpha prompted the suggestion that perhaps these amino acids were important for interacting with the positively charged nuclear localisation signal of the p65 subunit of NF- KB. Accordingly two regions, residues 284-286 and residues 300-302 (glutamic acid, aspartic acid, glutamic acid in both regions), were targeted for mutation (and referred to as the C-terminal mutants) to examine their role, if any, in IKBalpha -p65 association. A second set of mutants were generated following a protease sensitivity study on IKBalpha which revealed that residues 251 (tyrosine), 258 (tryptophan) and 275 (glutamic acid) were protected from digestion in the presence of p65. These amino acids were located in the low homology sixth ankyrin repeat of IKBalpha, thought to act as a flexible linker region between the highly conserved central five ankyrin repeats and the C-terminal region of the protein. Consequently, amino acids 258 and 275 were selected for mutation (referred to as the linker mutants). The in vitro characterisation of the C-terminal and linker mutants demonstrated that neither amino acids 258 and 275 nor amino acids 284-286 and 300-302 affected the ability of IKBalpha to interact with p65 homodimers, even under conditions of varying pH or ionic strength. However, residues 284-286 reduced the inhibitory capacity of IKBalpha with respect to p65 homodimers. The results indicated that the region of IKBalpha required for the inhibition of p65 DNA binding activity, possibly located in or around residues 284-286, was separable from the area of the protein responsible for association with p65. Attempts to phosphorylate the C-terminal and linker mutants in vitro using either casein kinase I or casein kinase II demonstrated that the C-terminal mutants were not efficiently phosphorylated by casein kinase II but were phosphorylated by casein kinase I. Both kinases were shown to phosphorylate wild-type IKBalpha and the linker mutants. Therefore, residues 284-286 and 300-302 were possibly important for the in vivo phosphorylation of IKBalpha through casein kinase II. The expression of the C-terminal and linker mutants from vectors transiently transfected into 293 cells revealed that residues 284-286 and residues 300-302 were required for either inducible or constitutive degradation of IKBalpha, but more likely constitutive turnover. All mutants appeared to undergo signal-induced ubiquitination. Furthermore, the mutants were capable of interacting with p65 and in a different cell line (Cos7 cells) all appeared to allow NF-KB-dependent transcription from a luciferase reporter. The final result was thought to be a consequence of different degradation characteristics existing within Cos7 cells compared to 293 cells. In the light of recent data, it was concluded that the C-terminal residues of IKBalpha were important for constitutive, rather than inducible turnover of IKBalpha.

A technique was developed for assaying amino acid: tRNA ligases extracted from tobacco XD cells grown in chemically defined medium (M-1D). The technique was based on the physiological enzymic reaction in which amino acid is aminoacylated to tRNA. tRNA was obtained from tobacco XD cells using a phenol extraction procedure. For two enzymes, arginine: tRNA ligase and leucine: tRNA ligase, assay conditions were optimised. Both enzymes had similar Km values for their cognate amino acids; were found to be unstable when stored at -10 and their activity was inhibited by ammonium sulphate and caesium chloride. During growth of tobacco XD cells, these two enzymes increased in activity. Amino acids appeared not to be involved in their regulation and attempts to perturb in vivo levels of aminoacyl-tRNA by use of amino acid analogues were unsuccessful. The use of the density labelling technique, which allows a distinction between pre-existing enzyme molecules and those that are newly synthesised, indicated that in M-ID both arginine; and leucine: tRNA ligases were synthesised de novo. Leucine: tRNA ligase was also degraded and therefore turned over as it increased in activity. The density labelling data did not allow a similar conclusion for arginine: tRNA ligase. During cell growth in nitrateless M-1D, there was no increase in the activity of arginine: and leucine: tRNA ligases, but both anzymes were found to be synthesised de novo. It was concluded, therefore, that they were both degraded and so turned over in nitrateless M-ID. Arginine: and leucine: tRNA ligases appeared to be synthesised from different amino acid pre-cursor pools and DEAE cellulose chromatography of enzyme extracts revealed the presence of three ligase species cognate for arginine but only two species cognate for leucine. The species cognate for arginine were in approximately equal proportions whereas one of the species cognate for leucine accounted for 80% of the total enzyme activity. The possibility that these multiple enzymic species might be responsible for the inability to demonstrate degradation of the arginine anzyme in M-1D was discussed. An accurate determination of the turnover rates of these/ these two enzymes could not be obtained due to the effects of re-cycling of total cell protein, but a comparison of turnover rates was attempted. The possible mode of regulation of these enzymes was discussed in relation to our observations and to those found in other systems.

No description available.

No description available.

The photosensitised oxidation of the conjugated diene ester methyl octadeca-9E,11E-dienoate gives an unsaturated cyclic peroxide (epidioxide) in high yield. This has been characterised spectroscopically. The 9,12-peroxide undergoes facile rearrangement to the 9,12-furanoid ester under a variety of reaction conditions. Catalytic reduction of the unsaturated peroxide cleaves the O-O bond. Bromination and epoxidation give dibromo and epoxy esters in high yield with the peroxide group still intact. Methyl 9(10)-hydroperoxyoctadec-10(8)E-enoates (1a,b), produced by photosensitised oxidation of methyl oleate are suitable substrates for the synthesis of substituted dioxolanes. Peroxymercuration of (1) affords on hydrogenodemercuration methyl 8,10- and 9,11-epdioxyoctadecanoates (2a,b) in good yield (45-70%). Bromodemercuration yields the corresponding bromo substituted cyclic peroxides (3a,b) in higher yield (95%). Direct bromination of the allylic hydroperoxides (1a,b) also affords the bromo substituted cyclic peroxides (3a,b) in almost quantitative yield, presumably via a bromonium ion intermediate. The photosensitised oxidation of methyl vernolate, ricinoleate, 12-oxooctadec-9Z-enoate and 12-bromooctadec-9Z-enoate leads to allylic hydroperoxides with a shifted double bond as required by the ene-mechanism. The products are thermally sensitive and sample lifetimes are short. The silver trifluoroacetate assisted reaction of alkyl halides with hydrogen peroxide has been investigated. Reaction with methyl 12-bromostearate has furnished for the first time methyl 12-hydroperoxystearate in 34% isolated yield. Reaction with methyl 12-bromooleate, however, is more complicated and leads to the formation of cyclopropane hydroperoxides via a homoallylic cation rearrangement, and to hydroperoxy-epidioxides presumably via methyl 12-hydroperoxyoleate which we were unable to isolate. Methyl 12-t-butylperoxyoleate has been produced by reaction of methyl 12-bromooleate with t-butylhydroperoxide in the presence of silver trifluoroacetate. None of these transformations occur if the silver salt is replaced by silver acetate. 13C nmr chemical shifts are reported for a number of oxygenated long chain aliphatic compounds, including, epoxides alcohols, hydroperoxides and cyclic peroxides. The influence of the oxygenated functional groups on the chemical shifts of neighbouring carbons have been determined and a comprehensive set of chemical shift parameters derived. Results are discussed where applicable in terms of steric and electric field effects.

1. The effect of pregnancy and lactation on some aspects of adipose tissue lipid metabolism in rat and mouse was studied. An attempt was made to elucidate control mechanisms for the changes in adipose tissue lipoprotein lipase activity in the rat during pregnancy and lactation. 2. Oxidation of glucose and synthesis of fatty acids from glucose by rat adipose tissue was either normal or elevated during early- and mid-pregnancy. Late pregnancy and lactation were characterized by low rates of glucose oxidation and fatty acid synthesis. 3. Rat adipose tissue lipoprotein lipase activity fell during early-pregnancy, prior to a recovery of enzyme activity in mid-pregnancy. The enzyme activity fell during late pregnancy to very low levels which were maintained until at least day 8 of lactation. Lipoprotein lipase activity in mouse adipose tissue fell during early-pregnancy and this low level of activity was maintained until day 17 of pregnancy, when an increase in activity occurred. The increased activity was maintained in early-lactation. 4. The response of rat adipose tissue in vitro to epinephrine stimulation in the release of FFA and glycerol was increased throughout pregnancy and early-lactation. Release of both FFA and glycerol was depressed on day 10 of lactation. Basal release of PPA increased but not significantly on days 7 and 12 of pregnancy and 3 of lactation. Glycerol release was elevated on day 12 of pregnancy and day 3 of lactation. Evidence is presented that the rate of FFA reesterification in the rat is decreased in early-to mid-pregnancy and increased in late pregnancy. Basal FFA and glycerol release in mouse adipose tissue in vitro were not significantly different from controls during pregnancy. FFA release was depressed on day 2 of lactation whereas glycerol release was increased. Both FFA and glycerol release in response to epinephrine stimulation increased, but not significantly, in late-pregnancy and markedly increased in early lactation. Prolactin injections had no significant effect on virgin rat adipose tissue lipoprotein lipase activity in vivo. Oestradiol benzoate markedly depressed lipoprotein lipase activity in virgin rat adipose tissue in vivo, and simultaneous administration of oestradiol benzoate plus prolactin did not decrease enzyme activity further. Simultaneous administration of oestradiol benzoate and a-ergocryptine produced a significant decrease in lipoprotein lipase activity in virgin rat adipose tissue in vivo. a-Ergocryptine administration to lactating rats reduced litter weight gain and increased the activity of lipoprotein lipase in adipose tissue in vivo.

1. β-Amylase was prepared from barley malt by salt extraction and purified by ammonium sulphate fractionation and gel filtration on Sephadex G-25 followed by chromatography on DEAE-cellulose. Traces of β-amylase activity were removed by low temperature storage under acid conditions. β-Amylase so prepared released no soluble blue dye from chromogenic alpha-1,4-glucans. 2. The kinetic parameters (Km and Vm) of β-amylase hydrolysing alpha-1,4-glucan polymers were found to increase in the presence of β-amylase. This was explained in relation to the chain length of the substrate. 3. Amyloses of different degree of polymerization (DP) were prepared by ethanol fractionation of thymol-precipitated amylose from soluble starch. The values of Km and Vm for β-amylase were shown to decrease with an increase in the DP of the substrate. The values of Km for the non-reducing terminal and of for the internal portions of the substrate were found to be 0.001mM and 0,00015mM, respectively. 4. Series of dialdehyde amyloses and borohydride-reduced dialdehyde amyloses were prepared. An explanation was given for the dependence of Km and Vm upon the degree of oxidation of dialdehyde and reduced dialdehyde substrates. 5. Inhibition studies involving maltose, dialdehyde and reduced dialdehyde amyloses were carried out. A theory to explain the inhibitory effect of oxidized amyloses upon β-amylase activity was put forward. 6. Immobilised β-amylase derivatives prepared using AE-cellulose and polyaminostyrene supports were found to retain 18% and 9.1%, respectively, of the original activity of the soluble enzyme. The effects of pH and temperature upon the immobilised derivatives was compared with the effects upon soluble β-amylase and the apparent value s of Km and Vm (Km' and Vm') for soluble starch and of Ki (Ki') maltose were determined for each preparation. The action patterns of the soluble enzyme and the immobilised derivatives on amylose were investigated by plotting the decrease in blue value of the amylose substrate against the increase in the reducing power of the solution as hydrolysis proceeded and were confirmed by chromatographic analysis of the reaction products and intermediates of maltoheptaose hydrolysis and explained in terms of the alteration of enzyme affinity towards the substrate upon immobilisation.