Christomanou H, Aignesberger A, Linke R P
Biol Chem Hoppe Seyler. 1986 Sep;367(9):879-90. doi: 10.1515/bchm3.1986.367.2.879.
Two nonenzymic activator proteins shown previously to strongly stimulate enzymic sphingomyelin degradation in vitro were purified from human Gaucher type 1 and control spleen. Activator A1 (molecular mass 6,500 Da) had affinity for ConA-Sepharose, while activator A2 (molecular mass 3,500 Da) did not. Monospecific antibodies to each activator protein were prepared in rabbits by immunization with protein purified from type 1 Gaucher spleen. A1 and A2 activators from Gaucher type 1 spleen were shown to be immunochemically identical to A1 and A2 activators from control spleen. However, A1 and A2 activators, whether isolated from Gaucher type 1 or control spleen, were shown to be distinct proteins. Immunochemical examination of all collected fractions during the purification revealed the existence of a third activator (molecular mass 6,000 Da), which was antigenically identical to A1 activator but had no affinity for ConA-Sepharose. The two forms of A1 activator showed similar mobility on immunoelectrophoresis differing from that of A2 activator. Fibroblast extracts from controls and patients with different variants of Gaucher disease were investigated using immunodiffusion against antisera to A1 or A2 activator. In contrast to normal and Gaucher (types 1, 2 and 3) cell extracts, those of a Gaucher patient with normal glucosylceramidase activity had no visible precipitin line towards the antiserum against the two forms of A1 activator. The lack of crossreacting material to antibodies against A1 activator was confirmed by radial immunodiffusion and rocket immunoelectrophoresis. A1 activator stimulated the basal glucosylceramidase activity 5-6 fold in fibroblasts from this patient, whereas the normal effect was only a 1.2-1.5-fold stimulation. The immunological results together with the biochemical data provide evidence for the lack of an activator protein in a variant form of human Gaucher disease for the first time.
先前已证明两种非酶激活蛋白可在体外强烈刺激酶促鞘磷脂降解，它们从人1型戈谢病脾脏和对照脾脏中纯化得到。激活剂A1（分子量6500道尔顿）对刀豆球蛋白A - 琼脂糖有亲和力，而激活剂A2（分子量3500道尔顿）则没有。通过用从1型戈谢病脾脏中纯化的蛋白质免疫兔子，制备了针对每种激活蛋白的单特异性抗体。结果表明，1型戈谢病脾脏中的A1和A2激活剂与对照脾脏中的A1和A2激活剂在免疫化学上相同。然而，无论是从1型戈谢病脾脏还是对照脾脏中分离得到的A1和A2激活剂，都显示为不同的蛋白质。纯化过程中对所有收集组分的免疫化学检查揭示了第三种激活剂（分子量6000道尔顿）的存在，它与A1激活剂在抗原性上相同，但对刀豆球蛋白A - 琼脂糖没有亲和力。两种形式的A1激活剂在免疫电泳上显示出相似的迁移率，与A2激活剂不同。使用针对A1或A2激活剂抗血清的免疫扩散法，研究了对照和成不同变体戈谢病患者的成纤维细胞提取物。与正常和戈谢病（1、2和3型）细胞提取物不同，一名具有正常葡萄糖脑苷脂酶活性的戈谢病患者的细胞提取物，在用针对两种形式A1激活剂的抗血清检测时，没有可见的沉淀线。通过放射免疫扩散和火箭免疫电泳证实了缺乏与针对A1激活剂抗体发生交叉反应的物质。A1激活剂使该患者成纤维细胞中的基础葡萄糖脑苷脂酶活性提高了5 - 6倍，而正常情况下的作用仅为1.2 - 1.5倍的刺激。免疫学结果与生化数据首次为人类戈谢病的一种变体形式中缺乏激活蛋白提供了证据。
