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Item The analysis of inquiry in students' conversations in the biochemistry laboratory : the elucidation of proton-coupled electron-transfer reaction mechanism in manganese superoxide dismutase through structural analysis of mutants : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand(Massey University, 2023) Hermawan, JatnikaSuperoxide dismutases (SODs) have very significant biological importance, protecting organisms against reactive oxygen species such as superoxide. They are also known as the fastest enzyme with the largest kcat/Km of any known enzyme. To perform super-fast enzymatic function, SOD must shuttle proton-coupled electrons in an efficient systematic way. However, since its discovery in 1968, the mechanistic nature of SOD catalytic function remains vague. Wide-ranging approaches have attempted to uncover the catalytic mechanism of the manganese-containing SOD, MnSOD, but there were experimental limitations that obstructed the investigations. Here, the structural analyses of two dimer interface mutants of MnSOD, S126D and S126W, explored possible changes in water structure near the active site providing new information to examine the hypothesis of the Glu170 bridge as a key player in the proton shuttle in the outer-sphere mechanism. To gain insight into the mechanism of the proton-coupled electron-transfer (PCET) reaction mechanism, the technique of single-crystal X-ray crystallography was used to observe the three-dimensional structure of Escherichia coli MnSOD mutants, analytical ultracentrifugation was used to observe quaternary association in solution, and protein stability was assessed by differential scanning calorimetry. The key residue Ser126 at the conserved but asymmetric dimer interface of the MnSOD was mutated with the initial intent to generate a monomeric species. Ser126 is not essential for activity and is not part of the active site, whereas Glu170 forms part of the dimer interface where Glu170 from one subunit forms part of the active site of the second subunit of the dimer. The loss of activity occurring in a monomeric MnSOD may indicate an alternative catalytic mechanism of the MnSOD enzyme. The substitution of Ser126 to Asp, intended to produce a monomeric species by charge repulsion, surprisingly produced a dimer at pH>7.5 with little change in structure at the Mn active site, but there was a 94 % reduction in catalytic activity. Partial loss of activity in Ec-MnSOD-S126D may be due to electrostatic effects of the negative charge ~7 Å from metal centre perturbing the Mnᴵᴵᴵ/Mnᴵᴵ redox couple. The substitution of Ser126 to Trp, intended to produce a monomeric species by steric bulk, enforces mostly monomeric Ec-MnSOD S126W in solution form, coupled with a 99.9 % reduction in catalytic activity. Here one mutation to a conserved dimer interface led to altered tertiary structure and a completely different dodecameric domain-swapped quaternary association in the crystalline state and complete loss of activity in Ec-MnSOD-S126W in the solution state. In the course of evolution, higher and less often lower degrees of oligomerisation have arisen. Evolving complexity does not require multiple mutations. As part of the scholarship requirements, this dissertation contains a pedagogical component. Student conversations in a guided inquiry third-year biochemistry laboratory were recorded and analysed to discover the extent of higher-order critical thinking that might occur. Although students initially struggled to move beyond core first-year laboratory skills, they were at all times strongly engaged in the project-style experiment, which ran over three five- to eight-hour sessions. Some progress in the level of inquiry was captured from their conversations from the first to the third laboratory session. A simple diagram and table were developed to help guide teachers in a guided inquiry-based learning in higher education.Item Aspects of the quantitative separation and estimation of thiamine and its phosphate esters : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1981) Scott, Paul NoelMethods for the separation and estimation of thiamine, thiamine monophosphate and thiamine diphosphate which would be applicable to biological extracts were investigated. Two methods for the estimation of thiamine were compared, the acid dye method and the thiochrome method. The thiochrome method was preferred as the acid dye method was more difficult to perform and some interference by certain amino acids was indicated. As both methods only estimate free thiamine, the optimum conditions for hydrolysis of thiamine phosphate esters by wheat germ acid phosphatase were also investigated. High phosphatase concentrations in the digestion mixture interfered with the extraction of thiochrome, by isobutanol, after oxidation of the free thiamine produced. Variation of the buffer in which the digestion was performed also affected the recoveries obtained. The inclusion of magnesium ions in the digestion mixture increased the activity of the enzyme so that it was possible to use an amount of phosphatase which was low enough to avoid interference with the extraction of thiochrome but which was sufficient to completely hydrolyse thiamine phosphate esters. The presence of magnesium ions also prevented the interference observed when formate rather than acetate buffers were used in the digestion mixture. A variety of separation techniques were investigated. Compared to paper and thin layer chromatography, high voltage paper electrophoresis (at 3kV in pH 3.5 buffer) gave the best and quickest separations. However only a 60% recovery was obtained after samples were eluted from the paper with 0.1M hydrochloric acid. Separation was achieved by elution of the thiochrome derivatives of thiamine, TMP and TDP from Sephadex G10 gel. Recoveries, estimated spectrophotometrically, indicated that this method could be used for the quantitative separation of thiamine and its phosphate esters. However since the method does not allow concentration of samples, it would be unsuitable for the estimation of biological extracts. Separation of thiamine and its esters using three ion exchange resins was also investigated. Partial separation of thiamine and its phosphate esters was obtained with Amberlite GC50 resin, the separation being determined by the form of the resin used. The hydrogen form of the resin allowed separation between TDP and thiamine-TMP while the sodium form separated thiamine from TMP-TDP. Neither form of the resin bound TDP firmly even when water was used as the eluent, so that separation of TDP and TTP would not be possible. Separation was attempted by eluting samples from Dowex 1-X8 resin with formate buffers of increasing ionic strength or pH. While the separation of thiamine, TMP and TDP appeared to be complete, by the elution profile, it was found that sample breakdown occurred. Electrophoresis of the eluted samples showed that the only peak which contained a single component was that corresponding to thiamine. Sample break-down was further indicated by a low recovery obtained when a sample containing only TDP was eluted. Identification of the peak contaminants was attempted using high voltage electrophoresis but proved difficult due to salt retardation affecting the positions of the peak components after electrophoresis. With Dowex 50 resin TDP and TMP were easily separated and eluted with ammonium acetate buffer of varying pH and ionic strength but the elution of thiamine required high pH or ionic strength solutions. Sample breakdown also appeared to occur on elution of samples from the resin. When TMP and TDP were eluted, separation appeared to be complete but a recovery of greater than 100% was obtained for TMP and both eluted compounds exhibited a progressive breakdown after elution. Sample breakdown was particularly notable when thiamine alone was eluted as 2 peaks were eluted and, after oxidation, yellow fluorescent material as well as the usual blue (characteristic of thiochrome) was observed. Characterisation of the yellow fluorescent compound(s) was attempted using electrophoresis, ultra-violet spectra and fluorescent spectra and it was found to be similar, but not identical, to thiamine.Item Histone H1 phosphorylation during mitosis : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2016) Bond, Sarah DHistone H1 phosphorylation is important for the regulation of high order chromosome organisation during mitosis. One of these phosphorylation sites in the linker histone subtype H1.4 is shown here to be phosphorylated by Aurora B kinase, a master regulator of mitosis. Altered phosphorylation of H1.4 on this phosphorylation site at serine 27 illustrated the significance of the timing of this phosphorylation. When serine 27 of H1.4 is mutated to prevent this phosphorylation chromosome congression to the equatorial plate during metaphase is hindered. In contrast, in the presence of the constitutive H1.4 serine 27 phosphorylation mimic, bridging and lagging chromosomes occurred, leading to a corresponding increase in the proportion of cells with a micronucleus. These phenotypes could be brought about through disruption of the Heterochromatin protein 1 family members bound to the adjacent methylated lysine. Such aberrations during mitosis can lead to genetic instability and ultimately aneuploidy, a hallmark of cancer. With the frequently reported over-expression of Aurora B in cancer this shows another mechanism in which this kinase, via histone H1.4 phosphorylation, can push a cell toward malignancy. Another important mitotic kinase, Cyclin dependent kinase 1 together with cyclin B, is responsible for the hyperphosphorylation of histone H1.4 during mitosis; which is required for condensing the cells genetic information into highly compact metaphase chromosomes. This vital mitotic event ensures the faithful transmission of the duplicated DNA into the dividing daughter cells. The mechanisms through which histone H1 hyperphosphorylation contribute to chromosome condensation are poorly understood. One mechanism through which this may occur is via the recruitment of condensation factors such as the condensins or Topoisomerase II. Here the interaction between the Condensin I subunit, CAPD2, and histone H1.4 is explored. CAPD2 interacts with the two most prominent linker histone subtypes, H1.4 and H1.2, through their C-terminal tails. H1.4 and CAPD2 can interact in vitro whilst each is phosphorylated by cyclin dependent kinase as they are during mitosis, in a manner dependent on RNA. Overall, these results indicate that histone H1.4 is a vital component of higher order chromatin and its phosphorylation is essential for the normal progression through mitosis.Item Characterisation of the conserved protein IMPACT from yeast (Yih1) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand(Massey University, 2012) Burr, Natalie SarahRegulation of translation under conditions of amino acid starvation is an important survival mechanism to ensure the continued viability of an organism. The accumulation of uncharged tRNA under amino acid starvation conditions triggers the activation of Gcn2, a kinase that phosphorylates the translation initiation factor eIF2a, inhibiting translation initiation. The protein IMPACT has been shown to inhibit Gcn2 by sequestering Gcn1, a protein that binds Gcn2 and is required for its function in vivo. IMPACT is a highly conserved protein, but despite its conservation, little is known about the role(s) it plays in the cell. The initial aim of this study was to investigate the three dimensional structure of Yeast IMPACT Homologue 1 (Yih1) using X-ray diffraction, in the hope that knowledge of the structure would inform further understanding of its many and varied complex biological functions. Because of the difficulties in obtaining diffraction quality crystals, a number of different techniques were employed that resulted in the production of a number of different plasmids for protein expression. These included surface entropy engineering, the use of folding and stability tags, and co-crystallisation with known binding partners. Further investigation into why the protein refused to crystallise revealed an innate heterogeneity that included a propensity to bind nucleic acids. Efforts were made to determine if this was related to function without success.Item Biosynthesis and metabolism of plant glycosides : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University(Massey University, 1968) Tapper, Brian AnthonyThe biosynthesis of selected cyanogenic glucosides and glucosinolates was examined in higher plants. Linen flax seedling shoots (linum usitatissimum L.) were used exclusively to study linamarin biosynthesis while prunasin biosynthesis was studies in both peach shoots (Prunus persica Batsch) and cherry laurel shoots (P. laurocerasus L.). Isoprppylglucosinolate and benzylglucosinolate were studied in scurvy grass seedlings (Cochlearia officinalis L.) and garden cress seedling shoots (Lepidum sativum L.) respectively. Altogether 9 isotopically labelled compounds were prepared as part of the study and 14 were administered to plant tissue. The quantity of cyanogenic glucosides was determined by measuring hydrogen cyanide following enzymic hydrolysis. The specific activity or dilution of the labelled compound after incorporation into the glucosides was determined and sued to judge the effectiveness of the administered compound as a precursor of the glucosides. Benzaldehyde from prunasin was measured as its semicarbazone and the isothicyanates. Obtained by enzyme hydrolysis of the glucosinolates, were identified by conversion to thiourea derivatives. Paper and thin layer chromatography and electrophoresis were used to separate non-volatile radioactive compounds. Isobutyraldoxime-U-14C, 2-oximinoisovaleric acid-U-14C, isobutyronitrile-1-14C and 2-hydroxyisobutyronitrile-1-14C were all incorporated into linamarin to extents comparable to that from L-valine-U-14c. By the use of 15N labelled compounds the C-N bond of isobutyraldoxime and 2-oximinoisovaleric acid was shown to remain intact during the cornversjon to linarnarin. Isobutyramide-1-14C and hydrogen cyanide-14c were not significantly incorporated into linamarin. Phenylacetaldoxime-U-14C, 2-oximino-3-phenylpropionic acid-2-14C and phenylacetonitrile-1-14c were converted to prunasin to greater extents than was L-phenylalanine-U-14C. Radioactivity from D,L-mandelonitrile-1-14C and, to a lesser extent, from hydrogen cyanide-14-C was also incorporated into the nitrile moiety of prunasin. Phenylacethydroxamic acid-1-14C was not significantly converted to prunasin. Linen 'lax seedling shoots were examined for both volatile and non-volatile intermediates. Radioactive precursors of linamarin were administered in the presence of other suspected intermediates or inhibitors of linamarin biosynthesis. Both isobutyraldoxime and isobutyronitrile were shown to be formed in the shoots from L-valine-U-14C. A non-volatile compound which accumulated in the presence of a few inhibitors Of linamaran biosynthesis was studied in detail. Treatment with acid under mild conditions yielded isobutyraldehyde while emulsin gave isobutyraldoxime. It was resistant to linamarase. Isobutyronitrile was a product of pyrolysis. The proposed structure for this compound is isobutyraldoxime-0-glucoside. Isobutyraldoxime-U-14C and phenylacetaldoxime-U-14C were both better precursors of the corresponding glucosinolates than were L-valine-U-14C or L-phenylalanine-U-14C. 2-Oximinoisovaleric acid-U-14C was not significantly incorporated into isopropylglucosinolate. It is concluded that aldoximes are intermediates in the biosynthesis of both cyanogenic glycosides and glucosinolates. Other intermediates proposed in cyanogenic glycoside biosynthesis are nitriles and 2-hydroxynitriles in that order, N-Hydroxyamino acids may be intermediates between amino acids and aldoximes. 2-Oximino acids may also be intermediates in cyanoglycoside biosynthesis although it is possible that the observed incorporation was by way of prior non-enzymic conversion to nitriles. The experiments with labelled administered compounds have outlined a pathway of cyanogenic glycoside biosynthesis which may now be profitably studied for confirmation at the enzymic level.Item Acetaldehyde metabolism in mammals : a thesis presented in partial fulfilment for the degree of Doctor of Philosophy in Biochemistry at Massey University, New Zealand(Massey University, 1975) Crow, Kathryn ElizabethThe metabolism of acetaldehyde in mammalian systems has been investigated both with in vitro studies on sheep liver aldehyde dehydrogenase, and by following changes in blood acetaldehyde levels in human volunteers. The intracellular localisation of aldehyde dehydrogenase was examined, using concurrent assays for marker enzymes. Average results for the distribution of the enzyme showed 30% of the total activity to be mitochondrial, 42% cytosolic, and 10% microsomal. The presence of activity in the microsomes was confirmed in studies where the enzyme was solubilised using sonication and Triton X-100 treatments. The cytoplasmic enzyme was purified by ammonium sulphate fractionations, ion exchange chromatography, and gel filtration, to a reproducible purity of 95%. Molecular weights of the native protein (205,000 - 220,000), subunit molecular weights (51,000 - 55,000) and behaviour during gel electrophoresis have been determined for both the cytoplasmic and mitochondrial sheep liver aldehyde dehydrogenases. An enzymic assay was developed for measuring very small amounts of acetaldehyde (down to 0.25 nmoles), using purified cytoplasmic sheep liver aldehyde dehydrogenase preparations. This assay has been applied to the determination of levels of acetaldehyde in the blood of human volunteers following ethanol ingestion. Untreated whole blood containing ethanol formed acetaldehyde, and the levels of acetaldehyde determined in either perchlorate-treated or untreated plasma were lower than those in whole blood. Acetaldehyde levels determined enzymically using perchloric acid supernatants of whole blood were reasonably close to the levels determined by gas chromatography, and the range of results (0.05 - 0.25 mg%) correlated well with similar results reported recently in the literature. A new ion exchange resin, DEAE Protion, was used during this investigation and separations of aldehyde dehydrogenase on forms of this resin and DEAE cellulose have been compared in an Appendix to the thesis.Item Enzyme promiscuity and the origins of cellular innovations : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Albany, New Zealand(Massey University, 2012) Soo, Valerie Wooi CheeBiochemistry textbooks define enzymes as being efficient and highly specific. However, these characteristics are usually associated with a lack of versatility, and therefore, an inability to evolve new functions. In spite of this, it is known that new enzymes can arise rapidly (such as when bacteria evolve antibiotic resistance). One hypothesis proposes that enzymes are actually promiscuous (Jensen, 1976); that is, they are able to carry out secondary reactions, in addition to the one they evolved to catalyze. The goal of this research was to explore the role that promiscuity plays in the origins and evolution of enzyme functions, using Escherichia coli as a model organism. In the first part of this thesis, I report the discovery of two enzymes (alanine racemase and cystathionine ß-lyase) that are reciprocally promiscuous, and are dependent on the cofactor pyridoxal 5’-phosphate (PLP) for activity. In vivo, the cofactor-mediated promiscuous activities of alanine racemase and cystathionine ß-lyase were each successfully improved to near wildtype levels using directed evolution experiments. These results extend Jensen’s hypothesis, and led me to propose that PLP played a significant role in the evolution of new enzymes, in the primordial world. In the second part of the thesis, I developed a comprehensive library-on-library screen to search for E. coli proteins that could mediate improved growth in environments containing either a foreign nutrient or a toxin. Proteins were over-expressed in an attempt to increase their weak, promiscuous activities, and to mimic the common genetic phenomenon of gene amplification. Over-expression of individual proteins conferred improved growth to the host cell in 35% of ~2,000 environments. The findings have important implications for understanding bacterial adaptation to new environments, such as when antibiotic resistance emerges. The ability of promiscuous proteins to drive the emergence of new phenotypes, when their expression is increased, validates the feasibility of the Innovation, Amplification and Divergence (IAD) model for the evolution of new genes (Bergthorsson et al., 2007). Overall, the work described in this thesis demonstrates that protein promiscuity is common, though difficult to predict a priori. My experimental results are consistent with the work of others, in suggesting that promiscuous activities are evolvable. Together, the high frequency and evolvability of promiscuous proteins appear to underpin many different cellular innovations.Item Genetic and biochemical studies on the urease enzyme system of Schizosaccharomyces pombe : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University(Massey University, 1993) Lubbers, Mark WilliamTwo indicator media were developed to detect urease activity in Schizosaccharomyces pombe colonies. These media were more sensitive than previously published media, permitted the rapid identification of urease mutants, were suitable for identifying urease positive transformants and were not affected by amino acid and nucleotide supplements. Four genes, designated ure1, ure2, ure3, and ure4, are required for urease activity in S. pombe. Each of the genes was mapped to an approximate genetic location by induced haploidization and meiotic recombination: ure1 on the left arm of chromosome III, 32 cM from fur1 and 50 cM from ade6; ure2 on the right arm of chromosome I, 69 cM from ura2 and 100 cM from ade4; ure3 on the right arm of chromosome I, 31 cM from ade4 and 91 cM from ura2; ure4 on the left arm of chromosome I, 100 cM from lys1. The lithium chloride method for S. pombe transformation was modified to improve the transformation frequency up to 100-fold by using carrier DNA and resuspending the cells in 0.9% NaCl after transformation. Urease mutants for each of the four ure genes were transformed with a S. pombe gene bank. Three different plasmid clones, each of which specifically complemented one of the ure1, ure3, or ure4 mutants, were isolated by complementation of the ure- phenotype. A gene bank clone complementing the ure2 mutant was not found. S. pombe urease was purified and characterized. The enzyme was intracellular and only one urease enzyme was detected by non-denaturing PAGE. The urease was purified 3,939-fold, with a 34% yield, by acetone precipitation, ammonium sulfate precipitation and DEAE-Sepharose ion exchange column chromatography. The native enzyme had Mr = 212,000 (Sepharose CL6B-200 gel filtration). One subunit was detected, with Mr = 102,000 (SDS-PAGE), indicating the undissociated enzyme contains two identical subunits. The specific activity was 709 μmol urea per min/mg protein. The enzyme was stable between pH 5.0 and pH 9.0. The optimum pH range for enzyme activity was pH 7.5 - pH 8.5. The Km for urea was 1.03 mM. The sequences of the amino-terminus and three tryptic peptides of the enzyme were determined: N-terminus - Met Gln Pro Arg Glu Leu His Lys Leu Thr Leu His Gln Leu Gly Ser, peptide T21 - Phe Ile Glu Thr Asn Glu Lys, peptide T40 - Leu Tyr Ala Pro Glu Asn Ser Pro Gly Phe Val Glu Val Leu Glu Gly Glu Ile Glu Leu Leu Pro Asn Leu Pro, peptide T43 - Glu Leu His Lys Leu Thr Leu His Gln Leu Gly Ser Leu Ala. The sequence of T43 overlaps the last 12 residues of the N-terminal sequence, extending the N-terminal sequence to 18 residues. The 18 N-terminal amino acids had 55.6% identity and 83.3% similarity (exact plus conservative replacements) with the jack bean urease N-terminal sequence. The seven amino acids of T21 had 42.9% identity and 100% similarity with the urease from Klebsiella aerogenes. Peptide T40 (25 amino acids) had only very poor identity with other sequenced ureases.Item Effects of ethanol on glycogen metabolism : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University(Massey University, 1997) Jeyarathan, PooranalingamThe effects of alcohol on glycogen structure and metabolism in fed, starved and starved-refed animals were studied in rats, taking into account factors such as post mortem degradation, careful isolation (native glycogen), and the separate structures and metabolism of low (cytosolic) and high (lysosomal) molecular weight glycogen. These studies were performed using the technique of density gradient ultracentrifugation. In fed animals, rats were administered doses of ethanol (intragastically) of either 2, 4, or 6 g/kg. The glycogen decreasing effect of ethanol was dose dependent. The lowest ethanol dose (2 g/kg) depleted liver glycogen content by 7-27%, while the highest dose (6 g/kg) showed 60-78% depletion. Ethanol doses of 4 g/kg and 6 g/kg decreased both low and high molecular weight glycogen almost evenly. There was slightly more low molecular weight glycogen loss than high with a 2 g/kg ethanol dose. In time course experiments, maximal glycogen depletion was observed at 90 minutes after an ethanol dose of 6 g/kg. After 24 hours, over-production of glycogen content was seen in ethanol treated rats. However, after 48 hours, liver glycogen content had returned to fed values in ethanol treated rats, although the content of low molecular weight glycogen was elevated relative to high molecular weight. Starvation of rats for 48 hours decreased both body weight and liver weight. The hepatic and skeletal muscle glycogen concentrations were decreased by 95% and 55% respectively. The livers of rats starved for 72 hours contained more liver glycogen than those starved for 24 hours and 48 hours. Ethanol accelerated glycogen degradation in the fed-to-starved transition. After 3 hours starvation, liver glycogen content had decreased to about half of the fed levels in ethanol treated rats. However, at 24 hours, glycogen content increased in the ethanol treated rats, to as much as twice that in the control animals. The rate and extent of depletion was greater in LMW glycogen than HMW glycogen at 6 hours and 12 hours. Studies on the effects of ethanol on the starved-to-refed transition were undertaken using two different protocols, chow refeeding and glucose administration by intragastric intubation. On chow refeeding after 48 hours starvation, liver glycogen repletion at 5 hours was decreased by about 30% in animals treated with ethanol dose of 4 g/kg. At longer time intervals there was no significant inhibition of glycogen resynthesis. The Inhibition of glycogen resynthesis at 5 hours was probably due both to a decrease in food intake in the treated animals and to inhibition of glycogen synthesis by ethanol. The rate and extent of resynthesis of high molecular weight glycogen was slower in treated rats than in control rats indicating that ethanol might preferentially inhibit the synthesis of high molecular weight glycogen, possibly through disruption or prevention of formation of disulphide bonds in the protein component of high molecular weight glycogen. Unlike liver, intragastric administration of 4 g/kg ethanol before chow refeeding following 48 hours starvation decreased muscle glycogen repletion until 24 hours refeeding, compared to the respective control rats. A single dose of intragastric administration of ethanol (3.45 g/kg) 1 hour before glucose refeeding by intragastric intubation decreased liver glycogen resynthesis by between 20-40% during the 2 hours after glucose administration. Ethanol probably delayed the peak reached in liver glycogen content by either decreasing glucose absorption, by inhibiting gluconeogenesis or glycogen synthesis, or a combination of all these factors. The overall effect of ethanol in inhibiting glycogen synthesis was not, however, nearly as great as that reported previously in similar experiments. In experiments where rats were given repeated doses of ethanol for 7 days, liver glycogen content was as much as 25 % higher in treated animals than in control animals at 24 and 48 hours after the last ethanol dose. Both low and high molecular weight glycogen had increased almost uniformly at 48 hours in the ethanol treated rats. Ethanol treatment had, however, decreased kidney glycogen content by 6-26% in the treated rats compared with the control rats, but the content of heart and muscle glycogen was not changed. The results of this research show that ethanol-induced overproduction of glycogen was seen in fed, fed-starved and starved-refed animals and also in repeated dose experiments. This finding is potentially of great importance in exercise physiology and sports science, in helping to develop recommendations for alcohol intake during training regimes.Item The effect of heat on the structure and aggregation behaviour of bovine B-lactoglobulins A, B and C : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University(Massey University, 1998) Manderson, Gavin AndrewThe bovine milk protein β-lactoglobulin (BLG) possesses a thiol group which becomes solvent exposed at elevated temperatures, leading to the formation of disulphide-linked milk protein aggregates. This phenomenon is of interest to the dairy industry because milk is heat-treated in many modern processes. This study is concerned with how the structure of BLG is altered during and as a consequence of heat treatment and how aggregates are formed. Bovine BLG exhibits genetic polymorphism and the A, B and C variants, present in New Zealand milks, differ in their susceptibilities to heat-induced structural change and aggregate formation, and their response to heat treatment is examined in the present study. This study used the following techniques: near and far UV CD, intrinsic protein fluorescence, hydrophobic probe fluorescence, thiol group solvent-exposure and both native-PAGE and SDS-PAGE. Spectroscopic and thiol exposure results suggest that the tertiary structure of BLG is altered during and as a consequence of heat treatment and that the amount of β-sheet in this protein does not alter appreciably as a consequence of heat treatment. PAGE results indicate that BLG forms a mixture of non-covalently-linked and disulphide-linked aggregates during heating, and that disulphide-linked dimers in particular are associated into larger non-covalently-linked aggregates. These non-covalently-linked aggregates are intermediates and large disulphide-linked aggregates are the end product of the BLG aggregation pathway. β-Lactoglobulin A forms aggregates, particularly large disulphide-linked aggregates, more slowly than BLGs B and C. Spectroscopic and thiol availability results suggest that the "intrinsic thermostability" of BLG C is appreciably greater than that of BLG B, which is slightly greater than that of BLG A. Furthermore, the extent of "irreversible structural change" in molecules of BLG C which occurs as a consequence of heat treatment is less than that in molecules of BLG A, which is less than that in molecules of BLG B. In the case of BLGs A and B this reflects the slower rate at which aggregates of BLG A form compared to that of BLG B. The present study has advanced the understanding of the BLG aggregation mechanism and how the A, B and C variants differ in their response to heat treatment.