Stinshoff K, Jatzkewitz H
Biochim Biophys Acta. 1975 Jan 23;377(1):126-38. doi: 10.1016/0005-2744(75)90293-4.
A cerebroside sulphatase (cerebroside-3-sulphate 3 sulphohydrolase, EC 3.1.6.8) assay based on radio thin-layer chromatography is described. The substrate was labelled by the catalytic addition of tritium to cerebroside sulphate. Using this assay the cerebroside sulphatase activity of sulphatase A (Aryl-sulphate sulphohydrolase, EC 3.1.6.1) from human liver and kidney in the absence of activators was investigated. The pH optimum of this reaction depends on the buffer concentration, being pH 4.5 at 50 mM and 5.3 at 10 mM sodium formate. With the latter concentration the apparent Km for cerebroside sulphate is 0.06 mM; SO2-4 and nitrocatechol sulphate inhibit noncompetitively with a Ki of 4.51 mM for Na2SO4 and 0.43 mM for nitrocatechol sulphate. The cerebroside sulphatase activity of sulphatase A is highly dependent on the ionic strength. The optimum sodium formate concentration is 10 mM, and the cerebroside suophatase activity decreases rapidly with increasing buffer concentration. The same concentration dependence is observed in the inhibitory effect of cerebroside sulphate on the arylsulphatase reaction. The inhibition decreases at increasing buffer concentrations, becoming an activation at 70 mM sodium formate. The progress curve of the cerebroside sulphatase reaction shows a deviation from linearity similar to that of the arylsulphatase reaction. Investigation of the effect of preincubation with cerebroside sulphate on the arylsulphatase activity of the enzyme shows that cerebroside sluphatase activity and inactivation of the enzyme by cerebroside sulphate occur simultaneously. These observations are interpreted as supporting the assumption that cerebroside suophate and arylsulphates are degraded at an identical active site on the same enzyme. Differences in the properties of the cerebroside sulphatase and the arylsulphatase reaction of the enzyme may be attributed to the differences in the physiocochemical state of the two substrates.
本文描述了一种基于放射性薄层层析的脑苷脂硫酸酯酶（脑苷脂-3-硫酸酯3-硫酸水解酶，EC 3.1.6.8）测定方法。底物通过将氚催化加成到脑苷脂硫酸酯上进行标记。使用该测定方法，研究了在无激活剂情况下人肝脏和肾脏中硫酸酯酶A（芳基硫酸酯硫酸水解酶，EC 3.1.6.1）的脑苷脂硫酸酯酶活性。该反应的最适pH取决于缓冲液浓度，在50 mM甲酸钠时为pH 4.5，在10 mM甲酸钠时为pH 5.3。在后一种浓度下，脑苷脂硫酸酯的表观Km为0.06 mM；SO42-和硝基邻苯二酚硫酸酯以非竞争性方式抑制，Na2SO4的Ki为4.51 mM，硝基邻苯二酚硫酸酯的Ki为0.43 mM。硫酸酯酶A的脑苷脂硫酸酯酶活性高度依赖于离子强度。最适甲酸钠浓度为10 mM，随着缓冲液浓度增加，脑苷脂硫酸酯酶活性迅速降低。在脑苷脂硫酸酯对芳基硫酸酯酶反应的抑制作用中也观察到相同的浓度依赖性。随着缓冲液浓度增加，抑制作用减弱，在70 mM甲酸钠时变为激活作用。脑苷脂硫酸酯酶反应的进程曲线显示出与芳基硫酸酯酶反应类似的偏离线性的情况。研究脑苷脂硫酸酯预孵育对该酶芳基硫酸酯酶活性的影响表明，脑苷脂硫酸酯酶活性和该酶被脑苷脂硫酸酯灭活同时发生。这些观察结果被解释为支持这样的假设，即脑苷脂硫酸酯和芳基硫酸酯在同一酶的相同活性位点上被降解。该酶的脑苷脂硫酸酯酶和芳基硫酸酯酶反应性质的差异可能归因于两种底物物理化学状态的差异。
