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Title: The role of tyrosine rich acidic matrix protein in the extracellular matrix
Author: Bear, Harriet Mary
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1997
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A number of purification strategies were performed in an attempt to produce highly purified, active TRAMP free from lysyl oxidase for fibrillogenesis and enzymatic studies. TRAMP was eventually purified by DEAE anion exchange followed by Superdex-75 size exclusion (in the presence of urea) and Mono Q FPLC anion exchange chromatography. Yields of 10μg of TRAMP per g wet weight starting material were obtained. TRAMP purified as above was active in fibrillogenesis assays using the 'warm start' technique, in buffers containing 30mM TES, 30mM Na2HPO4, 135mM NaCl pH 7.4. The acceleratory effect of TRAMP on collagen I fibril formation was also observed when the phosphate concentration was lowered to 10mM and when TES was removed. TRAMP has previously been shown to bind to in vitro reconstituted collagen fibrils if present during their formation. Reducing the phosphate concentration decreased the amount of TRAMP associated with collagen I fibrils in co-sedimentation assays, whilst subsequent removal of TES had no effect on TRAMP binding to collagen I fibrils. Preliminary observations also suggested that treatment of TRAMP with sulphatase had no effect on the ability of TRAMP to accelerate collagen I fibril formation. A solid phase assay showed TRAMP to bind collagen I monomers with a higher affinity than fibrils, suggesting a role for TRAMP in the early, nucleation phase of fibril formation. TRAMP was unable to reverse the inhibitory effect of decorin on fibril formation. The presence of decorin had no effect on TRAMP binding to collagen monomers in solid phase assays but led to an increase in the amount of TRAMP associated with collagen fibrils in co-sedimentation assays. Attempts to identify specific binding sites for TRAMP on in vitro reconstituted collagen I fibrils by immunogold labelling and electron microscopy were unsuccessful. Western blot analysis of murine tissues confirmed previous reports that TRAMP was present in lung and skeletal muscle and absent in brain and spleen.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available