The overall goal is to define the genes directing the synthesis of the mitochondrial enzyme complex, branched chain Alpha-ketoacid dehydrogenase [BCKD]. Inherited human mutations are known which specifically affect the function of BCKD known as Maple Syrup Urine Disease [MSUD]. These studies will increase our understanding of the defect and provide improved treatment for affected individuals. Immediate goals include: 1) Immunochemical characterization of BCKD proteins in human cells expressing defective function of the catalytic activity, 2) Biochemical characterization of the cytosolic synthesis and mitochondrial processing of the precursor proteins to BCKD, and 3) Construction of cDNA probes to study the genes and mRNAs directing the synthesis of these proteins. Polyclonal and monoclonal antibodies which specifically recognize the proteins of BCKD are used to screen mitochondrial proteins from fibroblasts derived from MSUD patients for cross-reactive material [CRM]. CRM- cells will be used to quantify BCKD proteins by crossed-immunoelectrophoretic methods. Western Blot analysis of two-dimensional gel separation of the mitochondrial proteins will also be used to study the mutant proteins. In vitro translation of RNA isolated from different sources, especially cells induced to overproduce mitochondrial proteins, will be used for the synthesis of these proteins. Processing studies will analysize conditions required for uptake of in vitro translated proteins by freshly isolated, respiratory competent mitochondria. Construction of cDNA probes involve using Lambdagt11 expression vectors with a human liver cDNA library to select clones producing the BCKD proteins. This involves to use of monospecific antibodies in selection of the clones and expansion of the selected clones in plasmid vectors.
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