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No description available.

Work on a wide variety of metabolic pathways indicates that these systems are often, if not always, organized as multienzyme complexes. Enzyme complexes have the potential to increase catalytic efficiency and provide unique mechanisms for the regulation of cellular metabolism. The flavonoid biosynthetic pathway of Arabidopsis is an excellent model for studying the organization, localization, and regulation of enzyme complexes at the cellular level. Flavonoids are specialized metabolites that perform many important physiological roles in plants. Protein interactions among several key flavonoid enzymes have been described. Moreover, at least two of the flavonoid enzymes have a dual cytoplasmic/nuclear localization. These results indicate that flavonoid enzymes assemble into one or more distinct complexes at different intracellular locations. The current study integrates a new technology, mass spectrometry, with well-established affinity chromatography methods to further characterize the organization and composition of the Arabidopsis flavonoid enzyme complex. One of the key flavonoid enzymes, chalcone isomerase (CHI), was used in these experiments to detect interacting enzymes. Recombinant thioredoxin (TRX), or TRX-CHI, was produced in E. coli, then purified by metal affinity chromatography, and covalently coupled to an activated resin, Affi-Gel 10. Extracts prepared from 4-day-old wild type or CHI-deficient lines of Arabidopsis were then passed over the column and the bound proteins eluted with sodium dedocyl sulfate (SDS). This eluate was then subjected to a liquid chromatography (LC) - mass spectrometry (MS) protocol developed for the analysis of complex peptide mixtures. An Agilent LC system coupled with an LTQ-MS instrument (Thermo Electron, San Jose, CA) was used for this purpose. Data analysis was performed with the Bioworks software package. Different optimization strategies were performed to improve the affinity chromatography, sample preparation, and the LC separation method. A novel approach has been developed for the MS analysis of biological samples containing contaminants such as salts and detergents. Protein extracts prepared from wild type Landsburg and mutant tt5(86) were analyzed by LC-MS/MS. A total of 491 proteins were identified for Landsburg and 633 for tt5(86) extracts using a combination of data filters and p-value sorting. All detected proteins had p<0.001 and most were identified by at least 2 unique peptides. / Master of Science

Humans have two plasminogen activators (PAs), tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), which generate plasmin to breakdown fibrin and other barriers to cell migration. Both PAs are used as pharmaceuticals but their efficacies are limited by their rapid clearance from the circulation, predominantly by parenchymal cells of the liver. At the commencement of the work presented here, the hepatic receptors responsible for mediating the catabolism of the PAs were little understood. tPA degradation by hepatic cell lines was known to depend on the formation of binary complexes with the major PA inhibitor, plasminogen activator inhibitor type-1 (PAI-1). Initial studies presented here established that uPA was catabolised in a fashion similar to tPA by the hepatoma cell line, HepG2. Other laboratories around this time found that the major receptor mediating the binding and endocytosis of the PAs is Low Density Lipoprotein Receptor-related Protein (LRP1). LRP1 is a giant 600 kDa protein that binds a range of structurally and functionally diverse ligands including, activated α2 macroglobulin, apolipoproteins, β amyloid precursor protein, and a number of serpin-enzymes complexes, including PA??PAI-1 complexes. Further studies for the work presented here centred on this receptor. By using radiolabelled binding assays, ligand blots, and Western blots on cultured cells, the major findings are that: (1) basal LRP1 expression on HepG2 is low compared to a clone termed, HepG2a16, but appears to increase in long term culture; (2) a soluble form of LRP1, which retains ligand-binding capacity, is present in human circulation; (3) soluble LRP1 is also present in cerebral spinal fluid where its role in neurological disorders such as Alzheimer??s disease is a developing area of interest; and (4) the release of LRP1 is a mechanism conserved in evolution, possibly as distantly as molluscs. The discovery, identification, and characterisation of soluble LRP1 introduces this protein in the human circulation, and presents a possible further level of regulation for its associated receptor system.