摘要
Mouse c-Abl type IV and human BCR/ABL proteins have been expressed in insect cells using the baculovirus system. The proteins were expressed as full-length polypeptides as judged by electrophoresis in denaturing gels. They were identified by immunoprecipitation and immunoblotting with antibodies against ABL peptides and, for BCR/ABL, against a BCR peptide. In these immunoprecipitates both proteins gave autophosphorylation principally on tyrosine. Both proteins were active tyrosine kinases, phosphorylating a variety of tyrosine-containing substrates. In fresh extracts both proteins contained phosphotyrosine as shown by Western blots with antiphosphotyrosine antibodies. Partial purification could be achieved readily using ion exchange columns, and the BCR/ABL protein, p210BCR/ABL, could be further purified to near-homogeneity using an antiphosphotyrosine column. Both enzymes required a divalent metal ion for activity. At low concentrations of ATP (2 μM) and with angiotensin II as substrate both enzymes were activated by Mn2+ or by Mg2+. No major differences in catalytic properties were found between the two isolated enzymes in solution. The oncogenic properties of p210BCR/ABL may be due to its different subcellular location, or to the presence of an intracellular inhibitor of c-Abl that does not inhibit BCR/ABL, or to altered substrate-specificity such that it can phosphorylate a unique substrate which is not recognised by c-Abl.
摘要译文
已经使用杆状病毒系统在昆虫细胞中表达了小鼠c-Abl IV型和人BCR / ABL蛋白。通过在变性凝胶中电泳判断，蛋白质表达为全长多肽。通过免疫沉淀和用抗ABL肽的抗体和BCR / ABL针对BCR肽的免疫印迹鉴定它们。在这些免疫沉淀物中，两种蛋白质主要在酪氨酸上产生自磷酸化。两种蛋白质都是活性酪氨酸激酶，磷酸化多种含酪氨酸的底物。在新鲜提取物中，两种蛋白质都含有磷酸酪氨酸，如蛋白质印迹所示，具有抗磷酸酪氨酸抗体。使用离子交换柱可以很容易地实现部分纯化，并且可以使用抗磷酸酪氨酸柱将BCR / ABL蛋白质p210 sup BCR / ABL / sup进一步纯化至接近同质性。两种酶都需要二价金属离子来进行活性。在低浓度的ATP（2μM）和血管紧张素II作为底物时，两种酶都被Mn sup 2 + / sup或Mg sup 2 + / sup激活。在溶液中两种分离的酶之间没有发现催化性质的主要差异。p210 sup BCR / ABL / sup的致癌特性可能是由于其不同的亚细胞定位，或者是由于存在不抑制BCR / ABL的c-Abl细胞内抑制剂，或者改变底物特异性，使其能够磷酸化c-Abl不识别的独特底物。
C.HughReynolds[a];Malcolm G.Willson[a];JohnGroffen[b];NoraHeisterkamp[b];Timothy C.Peakman[c];Martin J.Page[c];. Comparison of baculovirus-expressed c-Abl and BCR/ABL protein tyrosine kinases[J]. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1993,1181(2): 122-130