A new type of exo-beta-glucuronidase acting only on non-sulfated glycosaminoglycans.
Using chondroitin as a substrate, a new type of exo-beta-glucuronidase (EC 3.2.1.31) from rabbit liver was purified using a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephracryl S-300, affinity chromatography through heparin-Sepharose CL-6B, and preparative polyacrylamide gel electrophoresis. This enzyme acts only on non-sulfated glycosaminoglycans and their oligosaccharides and was shown to be quite different from exo-beta-glucuronidase, which does act on p-nitro-phenyl-beta-D-glucuronide with regard to the following properties. 1) Neither sulfated glycosaminoglycanoligosaccharides nor p-nitrophenyl-beta-D-glucuronide were substrates for the enzyme. 2) The molecular weight was found to be about 130,000 by gel filtration, compared with a molecular weight of 280,000-300,000 for beta-glucuronidase, which acts on p-nitro-phenyl-beta-D-glucuronide. 3) The enzyme showed maximal activity at pH 5.0, compared with an optimum pH of 4.5 for beta-glucuronidase, which acts on p-nitro-phenyl-beta-D-glucuronide. 4) The enzyme showed maximal activity in 0.075 M NaCl but no activity above 0.25 M NaCl. 5) The enzyme was inhibited strongly by compounds bearing a sulfate group. 6) The enzyme did not react with an antibody against beta-glucuronidase acting on p-nitrophenyl-D-glucuronide. It is suggested that the enzyme may be involved in the catabolism of glycosaminoglycans, acting especially on chondroitin after the desulfation reaction and/or hyaluronic acid, but showing little involvement with the detoxification system.
Nakamura
T
T
Department of Biochemistry, Hirosaki University School of Medicine, Japan.
Takagaki
K
K
Majima
M
M
Kimura
S
S
Kubo
K
K
Endoss
M
M
D000645:Ammonium Sulfate; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002807:Chondroitin / Q000378:metabolism; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D005966:Glucuronidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D005779:Immunodiffusion; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism
D006025:Glycosaminoglycans; D009844:Oligosaccharides; D013431:Sulfates; D012965:Sodium Chloride; D002807:Chondroitin; D005966:Glucuronidase; D000645:Ammonium Sulfate