Non-enzymatic modification by glucose of proteins induces protein damage through the formation of advanced glycation end products (AGEs). AGEs have been implicated in the pathogenesis of a number of diseases including diabetes. Pyridoxamine (PM) is an in vitro inhibitor of the formation of AGEs, which acts by blocking the conversion of the Amadori intermediate to AGEs. In both animal experiments and human clinical trials, PM has shown promising results in delaying the onset of diabetic renal disease. The goal of this study was to use model proteins to gain insight into how glucose modifications alter protein functionality and how PM prevents the loss of protein function. Biochemical analyses of ribonuclease, lysozyme, bovine serum albumin, and ubiquitin provided insight into the damaging effects of glucose modifications on protein function, which included the formation of the prominent AGE, carboxymethyllysine (CML), and the degradation and cross-linking of proteins. PM prevented CML formation, protected protein integrity, and preserved enzyme function. Mass spectrometry analyses show that in lysozyme residues K-96 and K-97 are more susceptible to CML formation, and PM protects against CML formation on these residues. Notably, K-96 and K-97 are in close proximity to the active site, suggesting that CML, but not Amadori, formation near the active site is detrimental to lysozyme activity. Also, PM was found to protect against the oxidation of tryptophan residues, W-62 and W-63. We suggest that the same mode of PM protection may also occur in vivo and may be responsible for PM efficacy.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03302006-150900 |
| Date | 12 April 2006 |
| Creators | Mathis, Missy Elaine |
| Contributors | Billy G. Hudson, David E. Ong |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03302006-150900/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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