Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Reactive, oxygen derived free radicals are detoxified by an enzymatic pathway RA-5 which includes the enzymes superoxide dismutase (CuZn and Mn SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). In an effort to understand the molecular evolution and antiquity of these enzymes, comparative techniques were developed for identification and visualization of protein family relationships. Analysis of amino acid sequence data indicated that these enzymes had expected phylogenetic relationships and that the two SOD enzymes were evolutionarily convergent. Multiple sequence alignments of the SOD, CAT and GSH-Px families of proteins were represented quantitatively and used to probe the Swiss-PROT database for proteins which shared similarity with previously identified, enzyme specific, conserved domains (e.g. active site). Several hundred distantly related sequences including proteins from viruses, prokaryotic and eukaryotic organisms were identified as similar to each of the enzymes under study. Further analysis of this data supports the "exon-shuffling" hypothesis which states proteins were assembled from small modular exons or domains (visible as ancient conserved regions in the present analysis) which were capable of recombination. In addition, comparative data derived from analysis of synonymous and nonsynonymous substitutions in these enzymes indicated that CuZn SOD, although part of a coordinated biochemical pathway, may be under separate selective pressures.;Biochemical assays indicated the expression of these enzymes, assessed as specific enzyme activity, was tissue-specific (in erythrocyte lysates, kidney, liver, and lung) and developmentally regulated in inbred mouse lines. In general, enzyme activities increased with age and were highest in the liver. The CuZn SOD and Mn SOD enzymes had different expression patterns: Mn SOD activity was low and unchanging or decreased while CuZn SOD activity increased with aging. Factor analysis of enzyme variation established their coordinated expression. Administration of xenobiotics such as ethanol modified the expression of these enzymes in a strain-specific manner. Segregation analysis of CAT activity phenotypes (among the most variable of the enzymes studied) in a number of genetic crosses that included reciprocal F{dollar}\rm\sb1s,{dollar} backcrosses, F{dollar}\sb2{dollar} and in some recombinant inbred lines allowed testing of mathematical models of inheritance. These studies indicated that multiple genetic factors/loci must govern the complex and tissue-specific expression of CAT in mice. Further studies will be needed to characterize these loci at the molecular level.



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