The overall objective of this research program is to elucidate the mechanisms which regulate the expression of complex macromolecules such as proteoglycans. The primary focus of the proposal is on a specific question of proteoglycan expression; how the machinery for the final processing step, sulfation, a common posttranslational modification of proteins, lipids and carbohydrates, and especially the proteoglycans, is organized and controlled in higher organisms. These studies are aided by the availability of a mutant model system which exhibits altered proteoglycan production, concomitant with abnormal growth and development, due to a defect in sulfation. The following specific aims are designed to advance the understanding of the sulfurylase/kinase complex from higher organisms structurally and functionally. Specifically, the relationships of the mammalian bifunctional enzyme and the individual sulfurylase and kinases from other sources will be compared mechanistically and molecularly. Murine sulfurylase/kinase has been cloned, sequenced and expressed and is now amenable for mutagenesis studies for biochemical, enzymatic and 3 D structural analysis. The specific molecular defect responsible for the brachymorphic phenotype will be elucidated. The PAPS translocase will be isolated and characterized, and used to reconstitute in vitro a functional PAPS activation transport utilization system that can be analyzed with respect to rate limiting steps, sites of regulation and nature and number of interacting components. The range of methodology necessary for accomplishing these goals includes: peptide purification and amino acid sequencing, cDNA cloning and sequencing, enzyme assay and kinetic analysis, organic synthesis of nucleotide analogs, cell and tissue culture and propagation of the rate inbred mouse strain. These studies have the long term goal of providing a model of the temporal and topological organization of this critically important pathway, how it is regulated and then to correlate defects in the overall pathway with abnormal growth and development.
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