Thomas G H, Raghavan S, Kolodny E H, Frisch A, Neufeld E F, O'Brien J S, Reynolds L W, Miller C S, Shapiro J, Kazazian H H, Heller R H
Pediatr Res. 1982 Mar;16(3):232-7. doi: 10.1203/00006450-198203000-00014.
Serum samples from two unrelated, clinically normal individuals lacked detectable hexosaminidase A by heat inactivation and electrophoretic analysis. In contrast, 15 and 17% of the hexosaminidase in their leukocytes and 23 and 26% of the hexosaminidase of their cultured fibroblasts had the heat stability and electrophoretic properties of the A form of this enzyme. An in vitro measurement of fibroblasts GM2 ganglioside-beta-galactosaminidase was in the range expected for Tay-Sachs disease (TSD) heterozygotes (2.5 and 3.1 versus a normal mean of 3.7). In contrast, fibroblasts from a patient with TSD, analyzed in an identical fashion, contained no detectable activity. Ten days after addition of labeled GM2 ganglioside to the medium of the cultured fibroblasts, 43 and 59% of the radioactivity taken up by the cells of these patients remained as unhydrolyzed ganglioside as compared with 94% in TSD fibroblasts and 42% in control cells. An analysis of sphingolipid composition by high performance liquid chromatography although the endogenous level of GM2 was elevated in TSD fibroblasts (0.39 nmoles/mg protein) there was no increase in the cells of these patients (0 and 0.12 versus control of 0.17 nmoles/mg protein). Finally, the synthesis of hexosaminidase was examined by an electrophoretic analysis of immunoprecipitates of the enzyme precursors that had been radiolabeled by culturing fibroblasts in medium containing [3H]-leucine. These studies revealed a normal pattern of biosynthesis, processing and secretion of the alpha and beta chains. The ratio of the alpha chain to the beta chain, however, was in the range expected for TSD heterozygotes.
通过热灭活和电泳分析，来自两名无亲缘关系、临床正常个体的血清样本中未检测到己糖胺酶A。相比之下，他们白细胞中15%和17%的己糖胺酶以及培养的成纤维细胞中23%和26%的己糖胺酶具有该酶A形式的热稳定性和电泳特性。对成纤维细胞GM2神经节苷脂-β-半乳糖胺酶的体外测量结果处于泰-萨克斯病（TSD）杂合子预期范围内（分别为2.5和3.1，正常平均值为3.7）。相比之下，以相同方式分析的一名TSD患者的成纤维细胞未检测到活性。在向培养的成纤维细胞培养基中添加标记的GM2神经节苷脂10天后，这些患者细胞摄取的放射性中有43%和59%仍为未水解的神经节苷脂，而TSD成纤维细胞中为94%，对照细胞中为42%。通过高效液相色谱法对鞘脂成分进行分析，尽管TSD成纤维细胞中GM2的内源性水平升高（0.39纳摩尔/毫克蛋白质），但这些患者的细胞中并未增加（分别为0和0.12，对照为0.17纳摩尔/毫克蛋白质）。最后，通过对在含有[3H]-亮氨酸的培养基中培养成纤维细胞而进行放射性标记的酶前体免疫沉淀物的电泳分析，检测了己糖胺酶的合成。这些研究揭示了α链和β链生物合成、加工和分泌的正常模式。然而，α链与β链的比例处于TSD杂合子预期范围内。
