The influence of polyphosphates upon the emulsification of enzymatic hydrolyzates of casein and lactalbumin and upon salt-soluble meat proteins was determined by a model system in which oil-in-water emulsions were formed. Sodium acid pyrophosphate, sodium tripolyphosphate and sodium hexametaphosphate were mixed together in a weight ratio of 4:2:4, respectively, to form a polyphosphate blend. This blend was added at a level of 0.5% (w/v) to casein or lactalbumin dissolved in 3% NaCl, pH 6.0. The polyphosphate blend was also added at the above level to meat proteins solubilized in 3% NaCl, pH 5.7. Emulsified volume (EV), total g of oil emulsified per 25 ml of protein solution, was determined for the above proteins with or without polyphosphates at varying protein concentrations. Data were also graphically expressed as emulsifying capacity (EC) and oil phase volume (OPV). For all proteins studied with or without polyphosphates, EV and OPV values increased with increasing protein concentration whereas EC values decreased. Addition of the polyphosphate blend to casein solutions containing protein levels in excess of 2 mg/ml resulted in significantly (P<0.01) higher EV levels than those of the controls. Conversely, the EV values of the polyphosphate-treated lactalbumin solutions were significantly (P< 0.01) lower than those of the controls at all protein levels tested. The diverse data obtained with these two proteins appear to be related to variations in molecular size and shape and to differences in the manner in which the proteinphosphate interactions occurred to cause the polyphosphates to enhance the emulsification of casein while depressing that of lactalbumin. Addition of the polyphosphate blend had little or no effect upon the emulsification of meat protein extracts obtained from fresh, frozen or refrozen samples. Thus, it was concluded that the polyphosphate blend did not modify nor exert any detectable influence upon the emulsification of solubilized meat proteins as tested in a model system. / Graduation date: 1975

In this research experiments were carried out to estimate hydrophobic and electrostatic interactions in soluble proteins. Five proteins, lysozyme, lactalbumin, ovalbumin, myoglobin and ribonuclease-A were chromatographed isocratically on a HIC column at several molalities (0.3-1.3m) of each of three different neutral salts, ferrous sulfate, ammonium sulfate and sodium sulfate. The calculated retention coefficients were then fitted to a recently developed chromatographic model in two ways. a) Multiple regression analysis was conducted to estimate C values according to the non-linear model (log k = A + B log m + C m) . b) Simple regression analysis was conducted to estimate C′ values according to the linear model (log k = A′ + C′m) at higher salt concentrations (above 0.3m). Results indicated that C′ values better estimate the hydrophobic interactions than C values, in experiments conducted only at higher salt concentrations. The comparison of C and C′ values with ANS, CPA, and Bigelow's average hydrophobicity indices showed no clear correlations. But, omission of ovalbumin improved the correlation coefficient of C′ with ANS. Both parameters indicated straight line relationships with molal surface tension increment of salts. Further, the same model was used to estimate the hydrophobic and electrostatic interactions in protein-protein interactions. Lysozyme and avidin were chromatographed on a lysozyme immobilized affinity column. Lysozyme-lysozyme interaction showed more affinity for hydrophobic interactions at low pH values. Avidin-lysozyme interaction showed both hydrophophobic and electrostatic interactions. Both interactions showed a greater change in the strength of hydrophobic interaction rather than the surface area of interaction, to changing pH. / Land and Food Systems, Faculty of / Graduate

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by Pui-Ki Kwan. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 134-143). / Acknowledgments --- p.i / Abstract --- p.ii / Abbreviations --- p.iii / Table of content --- p.v / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- The formation of body axes --- p.1 / Chapter 1.2 --- Maternal genes are essential for development --- p.8 / Chapter 1.3 --- Maternal gene bicoid is required for formation of anterior structures in the embryo --- p.9 / Chapter 1.4 --- Establishment of an anterior to posterior bcd protein gradient --- p.12 / Chapter 1.5 --- The bcd protein gradient regulates the downstream zygotic target genes in a concentration-dependent manner --- p.12 / Chapter 1.6 --- bcd protein acts as transcriptional regulators --- p.14 / Chapter 1.7 --- The anterior localization of bcd mRNA --- p.17 / Chapter 1.8 --- Components required for the localization of bcd mRNA --- p.17 / Chapter 1.8.1 --- Cis-acting elements --- p.17 / Chapter 1.8.1.1 --- BLE1 at 3' UTR directs localization of bcd mRNA --- p.19 / Chapter 1.8.2 --- Trans-acting elements --- p.21 / Chapter 1.8.2.1 --- "exuperantia, swallow and staufen are necessary for localization of bcd mRNA" --- p.21 / Chapter 1.8.2.2 --- exu protein is an absolute requirement for the localization --- p.24 / Chapter 1.8.2.3 --- Potential functions of exu based on the coding sequence --- p.25 / Chapter 1.8.2.4 --- Microtubules dependence of the localization --- p.26 / Chapter 1.8.2.5 --- Microtubules polarity directs localization of bcd RNAs --- p.27 / Chapter 1.9 --- Functions of exu in localization of bcd mRNA --- p.27 / Chapter CHAPTER 2 --- Characterization of deletion mutants of exu / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Construction of deletion mutants of exu --- p.31 / Chapter 2.2.1 --- Materials and Methods --- p.31 / Chapter 2.2.2 --- Results --- p.33 / Chapter 2.3 --- Analysis of exu protein in deletion mutants --- p.35 / Chapter 2.3.1 --- Materials and Methods --- p.35 / Chapter 2.3.1.1 --- Preparation of total ovary protein from the transgenic flies --- p.35 / Chapter 2.3.1.2 --- Analysis of protein content by SDS Polyacrylamide Gel Electrophoresis and immunoblotting --- p.35 / Chapter 2.3.2 --- Results --- p.36 / Chapter 2.4 --- Localization of bcd mRNA and exu protein in oogenesis --- p.39 / Chapter 2.4.1 --- Introduction --- p.39 / Chapter 2.4.2 --- Spatial and temporal distribution of exu protein in the deletion mutants --- p.41 / Chapter 2.4.2.1 --- Materials and Methods --- p.41 / Chapter 2.4.2.2 --- Results --- p.43 / Chapter 2.4.3 --- Spatial and temporal distribution of bcd mRNA in the deletion mutants --- p.56 / Chapter 2.4.3.1 --- Materials and Methods --- p.56 / Chapter 2.4.3.1.1 --- Principles of DIG-labeling and in situ hybridization --- p.56 / Chapter 2.4.3.1.2 --- Synthesis of DIG-labeled bcd DNA probe --- p.59 / Chapter 2.4.3.1.3 --- in situ hybridization of bcd mRNA in egg chambers using DIG-labeled DNA probe --- p.59 / Chapter 2.4.3.2 --- Results --- p.62 / Chapter 2.5 --- Discussion --- p.70 / Chapter CHAPTER 3 --- Determination of the interactions between exu and microtubules / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Localization of bcd mRNA and exu protein in the presence of drugs which destabilize cytoskeleton --- p.81 / Chapter 3.2.1 --- Materials and Methods --- p.81 / Chapter 3.2.2 --- Results --- p.82 / Chapter 3.3 --- Analysis of interactions between exu and microtubules by immunoprecipitation --- p.88 / Chapter 3.3.1 --- Materials and Methods --- p.88 / Chapter 3.3.1.1 --- Immunoprecipitation of exu protein and binding of microtubules --- p.88 / Chapter 3.3.1.2 --- Purification of tubulin from bovine or rat brains --- p.89 / Chapter 3.3.1.3 --- Determination of protein concentration of the tubulin stock by Folin-Lowry method --- p.90 / Chapter 3.3.1.4 --- Taxol-stabilized microtubules --- p.90 / Chapter 3.3.2 --- Results --- p.91 / Chapter 3.4 --- Analysis of interactions between exu and microtubules by cosedimentation --- p.94 / Chapter 3.4.1 --- Materials and Methods --- p.94 / Chapter 3.4.2 --- Results --- p.97 / Chapter 3.5 --- Analysis of interactions between exu and microtubules using detergent extracted ovary extract for co sedimentation --- p.100 / Chapter 3.5.1 --- Materials and Methods --- p.100 / Chapter 3.5.2 --- Results --- p.101 / Chapter 3.6 --- Analysis of intracellular distribution of exu protein and Release of exu protein by sodium carbonate treatment for cosedimentation with microtubules --- p.104 / Chapter 3.6.1 --- Materials and Methods --- p.104 / Chapter 3.6.1.1 --- Subcellular fractionation of ovary extracts --- p.104 / Chapter 3.6.1.2 --- Release of contents from fractions by sodium carbonate treatment --- p.105 / Chapter 3.6.1.3 --- Co sedimentation of exu protein with microtubules --- p.105 / Chapter 3.6.2 --- Results --- p.108 / Chapter 3.6.2.1 --- Intracellular distribution of exu protein --- p.108 / Chapter 3.6.2.2 --- Cosedimentation of exu protein with microtubules using Na2CO3 released extracts --- p.108 / Chapter 3.7 --- Cosedimentation of exu protein and microtubules in high ATP concentration --- p.113 / Chapter 3.7.1 --- Materials and Methods --- p.113 / Chapter 3.7.1.1 --- Preparation of ovary extracts and microtubules sedimentation --- p.113 / Chapter 3.7.1.2 --- Western blot using a chemiluminescent detection system --- p.114 / Chapter 3.7.2 --- Results --- p.115 / Chapter 3.8 --- Discussion --- p.122 / Chapter CHAPTER 4 --- Future Prospects --- p.125 / Appendix A Supplementary protocols --- p.126 / Appendix B Reagents --- p.131 / References --- p.134

No description available.

Bibliography: leaves [133]-151.

Includes bibliographical references. / 1 v. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1991

��-catenin is a multi functional protein that is involved in cell-cell adhesion and cell signaling. In non-stimulated cells, ��-catenin is tightly down-regulated by GSK-3��-dependent phosphorylation at Ser and Thr residues, followed by rapid ubiquitination and proteasomal degradation. It is well established that mutations within the regulatory GSK-3�� region lead to stabilized ��-catenin and constitutive ��-catenin/TCF-dependent gene activation. Furthermore, it has been shown that amino acids adjacent to codon 33, namely 32 and 34 of ��-catenin, are hotspots for substitution mutations in carcinogen-induced animal tumors. Thus, a major hypothesis of this thesis was that substitution mutations at codon 32 of ��-catenin interfere with phosphorylation and ubiquitination of ��-catenin. Site-directed mutagenesis was used to create defined ��-catenin mutants, namely D32G, D32N, and D32Y. The signaling potential of various ��-catenin was analyzed in a gene reporter assay by co-transfection with a hTcf cDNA with a reporter plasmid containing a Tcf-dependent promoter (TOPFlash). There was a significant enhancement of the reporter gene activity with all ��-catenin mutants compared to WT ��-catenin after 48 hours of transfection. Protein analysis by Western blotting showed massive accumulation of mutant ��-catenin. Antibody specific for phosphorylated ��-catenin showed that the accumulated D32G and D32N ��-catenin proteins were strongly phosphorylated both in vivo and in vitro, whereas D32Y ��-catenin exhibited significantly attenuated phosphorylation in vivo. Further studies showed, however, that none of the mutants was sufficiently ubiquitinated. In addition, inhibition of the proteasome activity by ALLN was associated with accumulation of cytosolic ��-catenin, which was transcriptionally inactive. This suppression of ��-catenin transcriptional capacity was independent of ALLN-associated apoptosis in the transfected cells. Furthermore, exogenous ��-catenin mediated modest cell survival and rendered cells sensitive to apoptotic stimuli. Thus, although codon 32 of ��-catenin is not a direct target for phosphorylation, results from this thesis suggested that it affects the phosphorylation and ubiquitination of the adjacent Ser-33 residue of ��-catenin, which is a direct target of GSK-3��. In addition, these results showed for the first time that the phosphorylation step of ��-catenin is not enough to regulate transcriptional activity, and that ��-catenin still needs to be ubiquitinated for successful down-regulation. / Graduation date: 2003

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