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|Title:||Structure and Metabolism of Heparin and Heparan Sulfate|
|Author(s):||Bienkowski, Michael Jerome|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Heparin was selectively cleaved at N-sulfoglucosamine residues by treatment with nitrous acid at pH 1.5. The oligosaccharide products, primarily disaccharides and tetrasaccharides, were then end-labeled by reduction with sodium borotritide and purified to homogeneity using a combination of BioGel P-2 chromatography and strong anion exchange HPLC. The unknown components of both the disaccharide and tetrasaccharide fractions were identified using a combination of chemical and enzymatic steps designed to convert each oligosaccharide to compounds of known structure. Following structure elucidation, the purified compounds were used a standards for the development of several HPLC based procedures in which all of the known structures could be identified and quantified in any heparin sample of interest.
In the second phase of this work, the procedures developed for heparin structural analysis were utilized to study the structure and metabolism of rat liver cell heparan sulfate. Monolayer cultures of rat liver cells incorporated radiolabeled sulfate and GlcN into heparan sulfate that was recovered in a culture medium (CM), extracellular matrix (Ma), and intracellular (IC) pool. Kinetic analysis of the secretion process showed that the CM heparan sulfate was continuously secreted and accumulated extracellularly. The cell surface or Ma heparan sulfate was also continuously secreted and delivered to the cell surface. In contrast to the CM heparan sulfate, the Ma material was continuously endocytosed and delivered to the lysosomes for degradation. Finally, the IC heparan sulfate pool was found to be a mixture of biosynthetic precursors of the CM and Ma pools, internalized Ma heparan sulfate undergoing degradation, and a pool that appeared to be destined to remain inside the cell.
Structural analysis of the polysaccharide chains isolated from the various pools indicated that they showed little structural variation. The chains were modified in a block-like fashion in that they contained unmodified sequences intermixed with regions of modified sequences. Furthermore, the modified sequences were clustered in regions of the polymer containing GlcNSO(,3) and these sequences contained virtually all of the IdoA and ester sulfates present in the intact polymer.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1984.
|Date Available in IDEALS:||2014-12-15|