Marchesini S, Viani P, Cestaro B, Gatt S
Department of Medical Chemistry and Biochemistry, University of Milan, Italy.
Biochim Biophys Acta. 1989 Mar 14;1002(1):14-9. doi: 10.1016/0005-2760(89)90058-1.
Two fluorescent derivatives of cerebroside sulfate ('sulfatide') have been synthesized and used as substrates for determining arylsulfatase A activity. These were 12-(1-pyrene)dodecanoyl cerebroside sulfate (P12-sulfatide) and 12(1-pyrenesulfonylamido)dodecanoyl cerebroside sulfate (PSA12-sulfatide). When incubated at pH 5.0 in the presence of 5 mM MnCl2 and 5.5 mM of taurodeoxycholate, either substrate was hydrolyzed by arylsulfatase A of human leukocytes. The rate of hydrolysis was proportional to the incubation time and concentration of enzyme; Michaelis-Menten type kinetics were observed with increasing concentrations of substrate. For determining the rate of hydrolysis, each of the two products (i.e., P12- and PSA12-cerebrosides) were separated from the bulk of respective unreacted sulfatide on small columns of DEAE-Sephadex A-25 and their fluorescence intensities read at 343-378 and 350-380 nm for the excitation and emission wavelengths for P12- and PSA12-cerebrosides, respectively. When extracts of skin fibroblasts derived from normal individuals and patients with Maroteaux-Lamy (lacking arylsulfatase B) or metachromatic leukodystrophy (lacking arylsulfatase A) were used as source of enzyme, P12-sulfatide was hydrolyzed by the former two but not by the latter cell extract. Several derivatives of cerebroside sulfate were also synthesized and found to inhibit the hydrolysis of pyrenesulfatide by leukocyte arylsulfatase A. The results demonstrate that these two pyrene containing sulfatides can be effectively used as specific substrates for the determination of arylsulfatase A activity in extract of cells and most probably also of tissues.
已合成了两种硫酸脑苷脂（“硫脂”）的荧光衍生物，并将其用作测定芳基硫酸酯酶A活性的底物。它们分别是12-(1-芘)十二烷酰硫酸脑苷脂（P12-硫脂）和12(1-芘磺酰胺基)十二烷酰硫酸脑苷脂（PSA12-硫脂）。当在pH 5.0、5 mM MnCl2和5.5 mM牛磺脱氧胆酸盐存在的条件下孵育时，两种底物均可被人白细胞的芳基硫酸酯酶A水解。水解速率与孵育时间和酶浓度成正比；随着底物浓度增加，呈现米氏动力学类型。为了测定水解速率，将两种产物（即P12-和PSA12-脑苷脂）在DEAE-葡聚糖A-25小柱上与大量未反应的各自硫脂分离，并分别在343 - 378 nm和350 - 380 nm读取P12-和PSA12-脑苷脂激发和发射波长下的荧光强度。当将来自正常个体以及患有马罗-拉米病（缺乏芳基硫酸酯酶B）或异染性脑白质营养不良（缺乏芳基硫酸酯酶A）患者的皮肤成纤维细胞提取物用作酶源时，P12-硫脂可被前两种细胞提取物水解，但不能被后一种细胞提取物水解。还合成了几种硫酸脑苷脂衍生物，发现它们可抑制白细胞芳基硫酸酯酶A对芘硫脂的水解。结果表明，这两种含芘硫脂可有效用作测定细胞提取物中芳基硫酸酯酶A活性的特异性底物，很可能也可用于测定组织中的该酶活性。
