Ludolph T, Paschke E, Glössl J, Kresse H
Biochem J. 1981 Mar 1;193(3):811-8. doi: 10.1042/bj1930811.
Enzymic cleavage of beta-N-acetylglucosamine residues of keratan sulphate was studied in vitro by using substrate a [3H]glucosamine-labelled desulphated keratan sulphate with N-acetylglucosamine residues at the non-reducing end. Both lysosomal beta-N-acetylhexosaminidases A and B are proposed to participate in the degradation of keratan sulphate on the basis of the following observations. Homogenates of fibroblasts from patients with Sandhoff disease, but not those from patients with Tay--Sachs disease, were unable to release significant amounts of N-acetyl[3H]glucosamine. On isoelectric focusing of beta-N-acetylhexosaminidase from human liver the peaks of keratan sulphate-degrading activity coincided with the activity towards p-nitrophenyl beta-N-acetylglucosaminide. A monospecific antibody against the human enzyme reacted with both enzyme forms and precipitated the keratan sulphate-degrading activity. Both isoenzymes had the same apparent Km of 4mM, but the B form was approximately twice as active as the A form when compared with the activity towards a chromogenic substrate. Differences were noted in the pH--activity profiles of both isoenzymes. Thermal inactivation of isoenzyme B was less pronounced towards the polymeric substrate than towards the p-nitrophenyl derivative.
通过使用底物a（一种在非还原端带有N-乙酰葡糖胺残基的[3H]葡糖胺标记的脱硫角质素硫酸酯），在体外研究了角质素硫酸酯的β-N-乙酰葡糖胺残基的酶促裂解。基于以下观察结果，推测溶酶体β-N-乙酰己糖胺酶A和B都参与角质素硫酸酯的降解。桑德霍夫病患者的成纤维细胞匀浆，但泰-萨克斯病患者的成纤维细胞匀浆则不能释放大量的N-乙酰[3H]葡糖胺。对人肝脏中的β-N-乙酰己糖胺酶进行等电聚焦时，角质素硫酸酯降解活性峰与对对硝基苯基β-N-乙酰葡糖胺的活性峰重合。一种针对人酶的单特异性抗体与两种酶形式都发生反应，并沉淀了角质素硫酸酯降解活性。两种同工酶的表观Km均为4mM，但与对显色底物的活性相比，B形式的活性约为A形式的两倍。两种同工酶的pH-活性曲线存在差异。同工酶B对聚合物底物的热失活比对硝基苯基衍生物的热失活不太明显。
