METABOLIC MONITORING OF GAG SYNTHESIS - TC3

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project is a part of a more general integrated technology project directed at biochemical and cellular characterization of glycosaminoglycan (GAG) function. GAGs are polymeric, differentially sulfated, carbohydrates forming an important part of the extracellular matrix of mammalian cells. Interactions of proteins with this matrix are important in directing cell migration, modulating cell signaling and regulating the physical properties of the extracellular fluids and structures. Characterizing the pathways by which GAGs are synthesized and modified, and developing methodology by which these pathways can be monitored in vitro and in vivo, is essential to understanding function. This, however, is not an easy task; the sulfation patterns of GAGs add extreme heterogeniety to the polymers, and degradation and isolation of simple products is not always an option. Ideally, methods that are sensitive to sufation position and applicable even in vivo would be developed. Nuclear magnetic resonance (NMR) provides one possible approach. Procedures for introducing NMR active isotopes (15N) into GAGs and using two dimensional 15N-1H spectra to characterize sulfation position and polymer types are therefore being developed. Also, methodology for the enhancement of signals to the level needed for in vivo monitoring of the metabolic processes leading to GAGs is being developed.