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    Heat-induced interactions of hemp protein particles formed by microfluidisation with β-lactoglobulin
    (Elsevier Ltd, 2024-07-01) Ma S; Acevedo-Fani A; Ye A; Singh H
    This study explored the effect of microfluidization on the dispersibility of hempseed protein (HP) and the interactions of microfluidised HP particles with β-lactoglobulin (β-lg) after heat treatment. Microfluidization increased the dispersible protein fraction from 10% (non-microfluidised) to a maximum of 58% (200 MPa, 6 passes) in HP dispersions. Dispersible HP particles were within the micro-sized range (d4,3 ≤ 2 μm) after microfluidization. Heat treatment (95 °C, 10–60 min) of HP particles with β-lactoglobulin (β-lg) induced protein association by sulphydryl-disulphide exchange reactions; β-lg association with HP particles initiated within the first 20 min. Additionally, the particle size (d4,3) values of co-heated HP particles with β-lg were significantly smaller than those found in HP particle dispersions heated alone, results that were in line with microscopy analysis. This suggests that β-lg could have restricted HP particle aggregation. In conclusion, combining microfluidization and heat treatment could offer a venue to modify the physical properties of plant/milk protein mixtures.
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    Investigation into the formation of a protein-protein complex between ATP sulfurylase and APS reductase in onion (Allium cepa L.) : a thesis presented in partial fulfilment of the requirements for the degree of Masters in Science in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2005) Cumming, Mathew Hoani
    This thesis has attempted to obtain evidence, both in vitro and in vivo, of complex formation between chloroplastidic isoforms of APS-reductase (APSR; EC 1.8.4.9) and ATP-sulfiirylase (ATPS; EC 2.7.7.4) of Allium cepa (onion). Genes encoding APSR and ATPS from onion were expressed in E. coli, and the recombinant proteins were expressed and purified with glutathione Sepharose 4B chromatography and ion exchange chromatography. SDS-PAGE. separation and Coomassie blue staining revealed an ATPS recombinant protein with a molecular mass of 50 kDa, and full length and a truncated form of APSR, with molecular masses of 55 kDa and 45 kDa. respectively. Three different approaches were used to investigate complex formation in vitro. Using an ELlSA-based technique, an association of recombinant ATPS with recombinant APSR was shown at pH 7.4, with a proposed 1:1 stoichiometry. However, when these ELISA experiments were conducted at pH 9.4, no evidence for complex formation was observed, suggesting that the complex is dependent on the pH of the buffered solution used. The second method was the ligand binding assay where recombinant protein APSR was immobilised onto PVDF membrane and then incubated at 25°C with a solution containing ATPS protein. The detection of bound ATPS was achieved using anti- ATPS IgG, and it was possible to detect a putative ATPS-APSR complex at pH 7.4, although the complex was unable to be detected at pH 7.8 or pH 9.4. The third method used was the immunoprecipitation assay, where anti-ATPS IgG that was conjugated onto Sepharose resin was used to precipitate recombinant ATPS (and any proteins bound to ATPS). The proteins precipitated were identified firstly by their molecular mass and subsequent western analysis with either biotinylated ATPS IgG or APSR IgG antibodies. Using this technique, recombinant APSR was able to be precipitated from solution using recombinant ATPS, at pH 7.4. To investigate the role of glutathione as a possible co-factor in the mediation of the protein complex, the tripeptide was added to the buffer used (final concentration between 0-10 mM) in the ELISA experiments. These experiments showed that glutathione had no effect on the formation of the complex. However investigation of the role of glutathione using the immunoprecipitation assay (added at 5.0 mM) demonstrated that glutathione did result in an increase in the amount of the ATPS-APSR complex. The immunoprecipitation technique was also used in attempt to isolate the complex in vivo from onion chloroplast extracts. However the ATPS-APSR complex was unable to be detected using this technique. In this thesis, the term in vivo is used to refer to experiments with chloroplast extracts and so will include ATPS, APSR and other proteins that may also contribute to any ATPS-APSR complex. The term in vitro refers to the direct interaction between recombinant ATPS and APSR only. To determine if the ATPS-APSR complex had any effect on the control of ATPS activity, the specific activity of ATPS was measured in vitro using recombinant ATPS and APSR. Results from these experiments demonstrated that the complex formation did not alter the activity of ATPS. A second technique was used to detect the ATPS-APSR complex in vivo. An ATPS affinity column, made with anti-ATPS IgG antibodies conjugated to Sepharose, was firstly incubated with recombinant ATPS, and the chloroplast extract from onion was then passed through the column and any bound proteins were eluted with solutions with high or low pH. Using this technique, APSR was unable to be identified as a protein that associated with ATPS, but a number of other proteins, with molecular masses of ca. 48 kDa, 45 kDa, 40 kDa and 28 kDa, were identified as being putative protein partners to ATPS. Another approach to signal whether ATPS complex formation with other protein partners (including APSR) has occurred is to examine changes in the kinetic properties of the enzyme. To do this, the Km values of ATPS for inorganic phosphate were determined in two cultivars of onions, grown hydroponically with varying sulfur supply. Measurements that were taken prior to and during bulbing showed that for at least one cultivar (Texas Grano) with sufficient sulfur, an increase in the K m value from 3.8 nM (prior to bulbing), to 0.15 µM (during bulbing) was observed. It is possible that this change in K m value is due to the formation of a protein-protein complex.
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    Identification of potential Gcn2 regulating proteins : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Albany, New Zealand
    (Massey University, 2016) Prescott, Hayley Dawn
    The viability of any organism relies greatly on their ability to adapt their metabolic processes in response to environmental stimuli. Proteins are essential for almost every intracellular biological process. Proteins are composed of amino acid precursors and the levels of amino acids within the cell available for protein synthesis requires careful monitoring. Amino acid conservation needs to be initiated in response to declining availability. Under conditions of amino acid deprivation, cessation of non-essential protein synthesis and activation of amino acid biosynthetic pathways is initiated. This regulatory mechanism is referred to as the General Amino Acid Control (GAAC) pathway. General control non-derepressible 2 (Gcn2) is a fundamental constituent of the GAAC response pathway. Gcn2 senses a decline in amino acid availability and initiates the stress response by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2a). The phosphorylation of eIF2a triggers a sequence of events resulting in increased translation of the transcriptional activator Gcn4, which subsequently induces selective expression of genes necessary for de novo amino acid synthesis. The activation and activity of Gcn2 is moderated by inhibitory and facilitative protein interactions. Published large scale purification studies identified many novel Gcn2 binding partners, some of which may function in Gcn2 regulation. However known Gcn2 regulators were absent from these datasets indicating that they were incomplete. This work aimed to identify and screen potential Gcn2 binding partners for those that regulate Gcn2 activity. Analysis identified 135 proteins that were potentially in complex with Gcn2. Of those, Sse1, Chs5, Ncl1, Tir4 and Npr1 were subsequently identified as potential Gcn2 regulators. For the purpose of comprehensively identifying novel Gcn2 binding proteins, a protocol was successfully optimised to enable Gcn2 affinity purification under conditions that would be specifically conducive for the maintenance of bonds between Gcn2 and its interaction partners. In this method, Gcn2 was overexpressed in cells to drive weak and/or transient interactions, and the usage of formaldehyde to crosslink interactions and further stabilise them was explored. This method will enable the future compilation of a comprehensive Gcn2 interactome.