Miura R O, Yamagata S, Miura Y, Harada T, Yamagata T
Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Anal Biochem. 1995 Mar 1;225(2):333-40. doi: 10.1006/abio.1995.1163.
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.
底物凝胶电泳可用于检测微量透明质酸酶（HAase）。在底物凝胶电泳中，透明质酸（HA）被包埋在凝胶中。为了检测其他糖胺聚糖（GAG）的降解酶，这些GAG的分子尺寸比HA小得多，我们开发了一种技术，通过在硫酸软骨素（CS）的还原端引入烯丙基进行化学修饰，以便将其固定在凝胶中。在有无十二烷基硫酸钠的情况下，在CS共聚凝胶上检测具有CS降解活性的酶。发现硫酸软骨素酶ABC和HAase的最小量分别为8微单位和0.35毫单位。通过使用包埋HA和修饰CS共聚凝胶的酶谱分析，已证明人血清HAase至少由两种具有各自底物特异性的同工型组成。使用这种方法，已证明子宫肿瘤组织分泌一种新型HAase，该酶在中性pH下可降解HA，但在任何pH下均不能降解CS。该方法也被证实适用于其他GAG，用于测定个体GAG降解酶。在未来的研究中，它将用于研究组织中每种GAG的调节。
