Kiefer M C, Schmid C, Waldvogel M, Schläpfer I, Futo E, Masiarz F R, Green K, Barr P J, Zapf J
Chiron Corporation, Emeryville, California 94608.
J Biol Chem. 1992 Jun 25;267(18):12692-9.
The insulin-like growth factor binding protein (IGFBP) family comprises six structurally distinct, but highly homologous proteins. They have been identified in serum and other biological fluids, tissue extracts, and cell culture media. We have recently cloned cDNAs encoding human IGFBP-4, -5, and -6 and have now expressed these BPs in yeast as ubiquitin (Ub)-IGFBP fusion proteins. Western ligand blotting with 125I-IGF II under nonreducing conditions of recombinant human (rh) IGFBP-containing yeast lysates revealed specific binding bands for IGFBP-4, -5, and -6 at apparent molecular masses of 24-26, 30-32, and 24-26 kDa, respectively, indicating processing of the fusion proteins. High-performance liquid chromatography-purified rhIGFBPs had virually the same amino acid composition, amino acid number, and NH2-terminal sequences as the native BPs. Except for the affinity of rhIGFBP-6 for IGF I (Ka = 8.5 x 10(8) M-1), the affinity constants of the three IGFBPs for IGF I and II lie between 1.7 and 3.3 x 10(10) M-1, i.e. 25-100 times higher than the IGF I and II affinities of the type I IGF receptor. When present in excess, rhIGFBP-4, -5, and -6 inhibited IGF I- and II-stimulated DNA and glycogen synthesis in human osteoblastic cells, but rhIGFBP-6 had only a weak inhibitory effect on IGF I in agreement with its relatively lower IGF I affinity constant. The results of this study show that the primary effect of the three rhIGFBPs is the attenuation of IGF activity and suggest that IGFBPs contribute to the control of IGF-mediated cell growth and metabolism.
胰岛素样生长因子结合蛋白（IGFBP）家族由六种结构不同但高度同源的蛋白质组成。它们已在血清和其他生物体液、组织提取物以及细胞培养基中被鉴定出来。我们最近克隆了编码人IGFBP - 4、- 5和- 6的cDNA，并且现已在酵母中作为泛素（Ub）- IGFBP融合蛋白表达了这些结合蛋白。在重组人（rh）含IGFBP的酵母裂解物的非还原条件下，用125I - IGF II进行的Western配体印迹显示，IGFBP - 4、- 5和- 6的特异性结合带分别出现在表观分子量为24 - 26 kDa、30 - 32 kDa和24 - 26 kDa处，表明融合蛋白发生了加工处理。高效液相色谱纯化的rhIGFBPs与天然结合蛋白具有几乎相同的氨基酸组成、氨基酸数量和NH2末端序列。除了rhIGFBP - 6对IGF I的亲和力（Ka = 8.5×10(8) M-1）外，这三种IGFBPs对IGF I和II的亲和力常数介于1.7和3.3×10(10) M-1之间，即比I型IGF受体对IGF I和II的亲和力高25 - 100倍。当过量存在时，rhIGFBP - 4、- 5和- 6抑制人成骨细胞中IGF I和II刺激的DNA和糖原合成，但rhIGFBP - 6对IGF I的抑制作用较弱，这与其相对较低的IGF I亲和力常数一致。本研究结果表明，三种rhIGFBPs的主要作用是减弱IGF活性，并提示IGFBPs有助于控制IGF介导的细胞生长和代谢。
