The long term objectives are to: determine the molecular basis of Menkes disease, elucidate the metabolic pathways of hepatic Cu utilization, determine how Cu is secreted into bile, and determine the regulatory mechanisms for Cu-protein synthesis. Hepatocytes isolated from the brindled mouse model of Menkes disease and nutritionally, Cu-deficient mice will be used to identify the defective Cu-accumulation component in liver. Developmental aspects will be examined with brindled and nutritionally, Cu-deficient neontes. Nutritionally, Cu-supplemented kidney cells and fibroblasts will be contrasted to the same cells from the brindled mice to determine if the primary defect is expressed in those cells which retain Cu. The time-dependent efflux of Cu from normal and Cu-deficient, isolated hepatocytes will be used to determine how Cu is secreted into bile which is relevant to Wilson's disease. In each case, column and electrophoretic profiles will be analyzed to identify cytosolic and particulate fraction components. Pulse-chase experiments will be used to delineate product-precursor relations. Cu-regulation of Cu/Zn-SOD synthesis in fungal cultures will be used as a model system for differentiating translational and transcriptional regulatory mechanisms. Genetic analyses of selected mouse strains will be used to determine the number of gene loci involved in the regulation of SOD protein synthesis. The approaches used in the fungal system will be extended to Cu-regulation of protein synthesis in mouse fibroblasts. Pathways leading to Cu incorporation into existing apo-proteins will be contrasted to Cu incorporation associated with protein synthesis.
