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|Title:||The Substrate Specificity of Testicular Hyaluronidase and Its Use in Sequencing Chondroitin Sulfates|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Testicular hyaluronidase was used to study the distribution of sulfated disaccharide units along chondroitin sulfate chains. The transglycosylation activity of the hyaluronidase was eliminated by use of excess levels of a second enzyme, (beta)-glucuronidase to remove all nonreducing terminal D-glucuronic acid residues from oligosaccharides present in the reaction mixture. This action prevented the oligosaccharides from participating in the transglycosylation reaction. Conditions were also designed to eliminate sulfatase activities present in the hyaluronidase preparation. the D-glucuronic acid released also provided a convenient assay of the hyaluronidase activity.
('35)SO(,4)-Labeled chondroitin sulfate was digested with hyaluronidase and (beta)-glucuronidase generating various sized oligosaccharide fragments. These were separated, analyzed and determined to contain hybrid sequences of 4- and 6-sulfated disaccharide units along the same oligosaccharide chain.
It was also observed that 4-sulfated sequences were being concentrated in the smaller oligosaccharides suggesting that they were being more readily degraded by the hyaluronidase. Kinetic studies at saturating levels of commercially obtained chondroitin 4- and 6-sulfates were performed. the rate of hydrolysis of the 4-sulfated isomer was 1.5 fold greater than that of the 6-sulfated isomer at pH 5.0. This difference was increased to 3 fold by raising the pH to 6.0, 5 fold by increasing the temperature to 45(DEGREES)C and finally as much as 10 fold by adjusting ionic strength parameters. To determine which sequences were actually being cleaved in these mixed substrate preparations methodologies were developed to determine the identity of the galactosamine residues which emerge on the reducing and nonreducing terminals of oligosaccharides generated early in the digestion. Increased concentrations of galactosamine-4-sulfate residues were found at both of these terminals but an absolute specificity of the hyaluronidase for cleavage of D-glucuronic acid residues adjacent to 4-sulfated moieties could not be concluded.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1982.
|Date Available in IDEALS:||2014-12-15|