Analysis of glycosaminoglycan-degrading enzymes by substrate gel electrophoresis (zymography).
Substrate gel electrophoresis is of use for detecting minute amounts of hyaluronidase (HAase). In substrate gel electrophoresis, hyaluronan (HA) is impregnated in a gel. To determine the presence of degradation enzymes for other glycosaminoglycans (GAGs), the sizes of whose molecules are much smaller than that of HA, we have developed a technique by which chondroitin sulfate (CS) is chemically modified by introducing an allyl group at the reducing end for its immobilization in the gel. Enzymes with CS-degrading activity were detected on a CS-copolymerized gel in the presence or absence of sodium dodecyl sulfate. The smallest amount of chondroitinase ABC and HAase was found to be 8 microU and 0.35 mU, respectively. By zymography using HA-impregnated and modified CS-copolymerized gels human serum HAase has been shown to consist of at least two isoforms each with its own substrate specificity. Using this method, uterine tumor tissue has been shown to secrete a novel HAase which degrades HA at neutral pH, but not CS at any pH. This method was also confirmed applicable to other GAGs for determining individual GAG-degrading enzymes. In future research, it will be used to examine the regulation of each GAG species in tissue.
Miura
R O
RO
Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Yamagata
S
S
Miura
Y
Y
Harada
T
T
Yamagata
T
T
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002809:Chondroitin Sulfates / Q000737:chemistry*; D002810:Chondroitinases and Chondroitin Lyases / Q000032:analysis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000032:analysis / Q000097:blood / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011133:Polysaccharide-Lyases / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity
D000178:Acrylamides; D006025:Glycosaminoglycans; D011108:Polymers; D020106:Acrylamide; D002809:Chondroitin Sulfates; D006821:Hyaluronoglucosaminidase; D002810:Chondroitinases and Chondroitin Lyases; D011133:Polysaccharide-Lyases