Abstract
An endo-beta-glucuronidase acting on chondroitin sulfate was isolated from rabbit liver and purified about 550-fold, using a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephacryl S-300, affinity chromatography through heparin-Sepharose CL-6B and preparative polyacrylamide gel electrophoresis. The pH optimum of this enzyme was 4.0 and the Km value 7 X 10(-3) M for chondroitin sulfate (Mr 40,000). The isoelectric point of the enzyme was found to be at pH 5.4. The molecular weight, estimated by gel filtration through Sephacryl S-200 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 35,000. This enzyme, which was found in the liver, kidney, spleen, and lung, hydrolyzed the glucuronyl galactose linkage of the linkage region of chondroitin sulfate possessing a very small peptide segment. The enzyme did not hydrolyze proteoglycan. It was concluded that an endo-beta-glucuronidase is involved in the catabolism of proteoglycan chondroitin sulfates.
Highlights
The bean p”N-acetylhexosaminidaseE, scherichia coli @-galactosidasea, nd Dactylium dendroides galactose oxidase were purchased from Sigma
Pronase P from Streptomyces griseus was obtained from Kaken glycosaminoglycan chains maybe degraded byexoglycosi- Kagaku Co., Tokyo, Japan
ADi-OS)were purchased from Seikagaku Kogyo Co., Tokyo, Japan
Summary
Chemical Analysis-Total hexuronic acid was determined by the method of Bitter and Muir [15].Free hexuronic acid and thereducing terminal hexuronic acid of glycosaminoglycans weredetermined by a termini. The costs of 6S, 2-acetamido-2-deoxy-3-O-(~-~-gluco-4-enepyranosyluarcoidn)i-c publication of this article were defrayed in part by the payment of 6-0-sulfo-D-galactopyranoseA; Di-OS, 2-acetamido-2-deoxy-3-0-(@-. This article must be hereby marked “aduer- D-g~uco-4-enepyranosy~uroniacid)-D-galactopyranose.All sugars tisement” in accordancewith 18U.S.C. Section 1734solely to indicate mentioned in this paper are of D configuration except for iduronic this fact. Protein was determined by the method of Lowry et al [18]using bovine serum albumin as a standard
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