Two-Dimensional Gel Electrophoretic Analysis of Cultured Human Fibroblasts Deficient in Galactokinase and Galactose-1-Phosphate Uridyl Transferase
Johnson, Kevin Marke
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The purpose of this project was to determine whether two-dimensional electrophoresis could be used to assay for galactose pathway enzyme deficiencies and to further the understanding of the molecular basis of galactosemia. The metabolism of galactose to glucose is controlled by a series of four enzymes in the Leloir pathway. Two clinical disorders are associated with the deficiency of the first two enzymes in this pathway: galactokinase deficiency galactosemia and galactose-l-phosphate uridyl transferase deficiency galactosemia. To validate the experimental approach of identifying and mapping proteins in a complex mixture, purified galactose pathway enzymes from yeast were characterized according to their charge, mass, and color on two-dimensional electrophoresis and compared to the complex pattern of whole yeast cell proteins. Mapping UDP galactose-4-epimerase was accomplished in the yeast extracts and a tentative identification of galactose-1-phosphate uridyl transferase was made. Excellent reproducibility between gels was demonstrated. Since purified human enzymes were not available for mapping, we utilized established molecular weight values from the literature and compared the appropriate regions of two-dimensional protein patterns of human fibroblasts to search for variant proteins that could be the genetic explanation for the clinical syndrome.of galactosemia. In summary, application of two-dimensional gel electrophoresis technologies to analyze fibroblasts from two galactosemic families resulted in our finding provisional genetic markers for galactokinase deficiency galactosemia and. galactose-1-phosphate uridyl transferase deficiency galactosemia in the human.With honors.