Blat C, Delbe J, Villaudy J, Chatelain G, Golde A, Harel L
Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
J Biol Chem. 1989 Jul 25;264(21):12449-54.
From medium conditioned by 3T3 cells, we had previously purified to apparent homogeneity a novel inhibitory diffusible factor of 45 kDa (IDF45), and then determined the amino-terminal sequence. IDF45 prevented reversibly the growth of chick embryo fibroblast (CEF). In these cells, DNA synthesis stimulated by 1% serum was 50% inhibited in the presence of 45 ng/ml (1 nM) IDF45. In the present article, we show that, in CEF, DNA synthesis stimulated by IGF-I was 100% inhibited in the presence of purified IDF45. Furthermore, the 45-kDa protein (IDF45) was, after Western blotting, able to bind IGF-I. The inhibitory effect of IDF45 upon serum stimulation did not seem to be the result of its inhibitory activity upon IGF-I stimulation, since stimulation by IGF-I and serum were additive. Moreover, it was possible to dissociate the two inhibitory effects: when added to v-src transformed CEF, IDF45 was able to 100% inhibit stimulation induced by IGF-I and was unable to significantly decrease stimulation induced by serum, as was previously observed. Taken together, our results strongly suggest that IDF45 has two distinct functions, one of which was to bind IGF-I and the other to inhibit serum stimulation. Indeed, it was impossible to separate the two functions when IDF45 was purified by cation exchange fast protein liquid chromatography, a method very different from reverse-phase fast protein liquid chromatography previously used for purification to apparent homogeneity of IDF45. On the other hand, if the IGF binding activity and inhibitory activity effect upon serum stimulation were carried by two different proteins, the presence of IGF-I (in conditions where most of the 45-kDa proteins were bound to IGF-I) should not have affected the activity of the molecule inhibiting serum stimulation. However, we observed the contrary: when IDF45 was bound to IGF-I, it lost its inhibitory effect upon stimulation induced by serum. This suggests that the two activities occurred on the same protein and that IDF45 is a bifunctional protein.
我们先前已从3T3细胞条件培养基中纯化出一种表观均一的45 kDa新型抑制性扩散因子（IDF45），并测定了其氨基末端序列。IDF45可可逆地抑制鸡胚成纤维细胞（CEF）的生长。在这些细胞中，在存在45 ng/ml（1 nM）IDF45的情况下，1%血清刺激的DNA合成被抑制了50%。在本文中，我们表明，在CEF中，在存在纯化的IDF45的情况下，IGF-I刺激的DNA合成被100%抑制。此外，经蛋白质免疫印迹法检测，45 kDa蛋白（IDF45）能够结合IGF-I。IDF45对血清刺激的抑制作用似乎不是其对IGF-I刺激的抑制活性的结果，因为IGF-I和血清的刺激作用是相加的。此外，有可能将这两种抑制作用分开：当添加到v-src转化的CEF中时，IDF45能够100%抑制IGF-I诱导的刺激，并且不能像先前观察到的那样显著降低血清诱导的刺激。综上所述，我们的结果强烈表明IDF45具有两种不同的功能，其中之一是结合IGF-I，另一个是抑制血清刺激。实际上，当通过阳离子交换快速蛋白质液相色谱法纯化IDF45时，不可能将这两种功能分开，这种方法与先前用于将IDF45纯化至表观均一性的反相快速蛋白质液相色谱法有很大不同。另一方面，如果IGF结合活性和对血清刺激的抑制活性作用由两种不同的蛋白质承担，那么IGF-I的存在（在大多数45 kDa蛋白与IGF-I结合的条件下）不应影响抑制血清刺激的分子的活性。然而，我们观察到相反的情况：当IDF45与IGF-I结合时，它失去了对血清诱导刺激的抑制作用。这表明这两种活性发生在同一蛋白质上，并且IDF45是一种双功能蛋白。
