Moghaddam A, Bicknell R
Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, U.K.
Biochemistry. 1992 Dec 8;31(48):12141-6. doi: 10.1021/bi00163a024.
Platelet-derived endothelial cell growth factor (PD-ECGF) has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The fusion protein was purified by one-step affinity chromatography on glutathione-agarose beads, and recombinant PD-ECGF was proteolytically cleaved with thrombin from its GST leader peptide to yield pure protein. Recombinant PD-ECGF stimulated [3H]methylthymidine uptake by endothelial cells in vitro; however, we were unable to detect stimulation of cell proliferation under a wide variety of conditions. We confirm that in accord with the recent report that PD-ECGF and human thymidine phosphorylase are products of the same gene [Furukawa, T., Yoshimura, A., Sumizawa, T., Haraguchi, M., & Akiyama, S. I. (1992) Nature 356, 668] recombinant PD-ECGF has thymidine phosphorylase activity comparable to that of E. coli thymidine phosphorylase. Further, E. coli thymidine phosphorylase was able to mimic the activity of recombinant PD-ECGF in the [3H]methylthymidine uptake assay, and it appears that recombinant PD-ECGF's effect on the uptake of thymidine by endothelial cells may be due to modulation of cellular thymidine pools. The mechanism by which PD-ECGF stimulates angiogenesis remains to be elucidated.
血小板衍生的内皮细胞生长因子（PD - ECGF）已在大肠杆菌中作为与谷胱甘肽S - 转移酶（GST）的融合蛋白表达。该融合蛋白通过在谷胱甘肽琼脂糖珠上的一步亲和层析进行纯化，并且重组PD - ECGF用凝血酶从其GST前导肽上进行蛋白水解切割以产生纯蛋白。重组PD - ECGF在体外刺激内皮细胞摄取[³H]甲基胸苷；然而，在多种条件下我们未能检测到对细胞增殖的刺激作用。我们证实，与最近的报道一致，即PD - ECGF和人胸苷磷酸化酶是同一基因的产物[古川，T.，吉村，A.，住泽，T.，原口，M.，&秋山，S. I.（1992）《自然》356，668]，重组PD - ECGF具有与大肠杆菌胸苷磷酸化酶相当的胸苷磷酸化酶活性。此外，大肠杆菌胸苷磷酸化酶在[³H]甲基胸苷摄取试验中能够模拟重组PD - ECGF的活性，并且似乎重组PD - ECGF对内皮细胞摄取胸苷的作用可能是由于细胞内胸苷池的调节。PD - ECGF刺激血管生成的机制仍有待阐明。
