Hall S W, LaMarre J, Marshall L B, Hayes M A, Gonias S L
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
Biochem J. 1992 Jan 15;281 ( Pt 2)(Pt 2):569-75. doi: 10.1042/bj2810569.
The binding of 125I-labelled transforming growth factor-beta 1 (TGF-beta 1) to human alpha 2-macroglobulin (alpha 2M) was studied by native PAGE and autoradiography. TGF-beta 1 bound preferentially to alpha 2M-methylamine and minimally, if at all, to native alpha 2M. Preparations of alpha 2M-proteinase complex were generated by incubating a standard concentration of alpha 2M (0.4 microM) with different concentrations of trypsin, chymotrypsin or neutrophil elastase (0.04-2.0 microM). The 125I-TGF-beta 1-binding activity depended on the initial ratio of active proteinase to alpha 2M, or r value, used to form the alpha 2M-proteinase complex. With all three proteinases, r values of 2 or greater yielded preparations with unchanged or decreased TGF-beta 1-binding activity relative to native alpha 2M. By contrast, r values near 1 yielded preparations with significantly increased TGF-beta 1-binding activity. The results of [3H]thymidine-incorporation studies performed in mouse keratinocytes were consistent with the 125I-TGF-beta-binding experiments. alpha 2M-trypsin and alpha 2M-chymotrypsin prepared at an r value of 1.0 counteracted the activity of TGF-beta 1, whereas the equivalent complexes prepared at an r value of 3.0 had no effect. As determined by SDS/PAGE, 125I-TGF-beta 1 binding to alpha 2M-methylamine was at least 80% non-covalent. Reaction of alpha 2M-methylamine with iodoacetamide or 5,5'-dithiobis-(2-nitrobenzoic acid) decreased the percentage of covalent binding but had no effect on total binding. Neuraminidase treatment had no effect on the binding of 125I-TGF-beta 1 to alpha 2M-methylamine. Cleavage of the 'bait regions' in alpha 2M-methylamine by prolonged treatment with trypsin also had no effect. These studies suggest that TGF-beta 1 binding to alpha 2M is enhanced by conformational change in the proteinase inhibitor resulting from reaction with proteinase or amine. If both proteinase-binding sites in a single alpha 2M molecule are occupied, TGF-beta 1-binding activity is decreased or perhaps eliminated.
通过非变性聚丙烯酰胺凝胶电泳（native PAGE）和放射自显影技术研究了125I标记的转化生长因子-β1（TGF-β1）与人α2-巨球蛋白（α2M）的结合情况。TGF-β1优先与α2M-甲胺结合，与天然α2M的结合极少（若有结合的话）。通过将标准浓度的α2M（0.4微摩尔）与不同浓度的胰蛋白酶、糜蛋白酶或中性粒细胞弹性蛋白酶（0.04 - 2.0微摩尔）孵育来制备α2M-蛋白酶复合物。125I-TGF-β1的结合活性取决于用于形成α2M-蛋白酶复合物的活性蛋白酶与α2M的初始比例，即r值。对于所有这三种蛋白酶，r值为2或更大时所制备的复合物相对于天然α2M，其TGF-β1结合活性不变或降低。相比之下，r值接近1时所制备的复合物具有显著增强的TGF-β1结合活性。在小鼠角质形成细胞中进行的[3H]胸腺嘧啶核苷掺入研究结果与125I-TGF-β结合实验一致。以r值1.0制备的α2M-胰蛋白酶和α2M-糜蛋白酶可抵消TGF-β1的活性，而以r值3.0制备的同等复合物则无此作用。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）测定，125I-TGF-β1与α2M-甲胺的结合至少80%是非共价的。α2M-甲胺与碘乙酰胺或5,5'-二硫代双（2-硝基苯甲酸）反应会降低共价结合的百分比，但对总结合无影响。神经氨酸酶处理对125I-TGF-β1与α2M-甲胺的结合无影响。用胰蛋白酶长时间处理α2M-甲胺以切割其“诱饵区”也无影响。这些研究表明，由于与蛋白酶或胺反应导致蛋白酶抑制剂构象改变，增强了TGF-β1与α2M的结合。如果单个α2M分子中的两个蛋白酶结合位点都被占据，TGF-β1结合活性会降低或可能消除。
