Lydon N B, Gay B, Mett H, Murray B, Liebetanz J, Gutzwiller A, Piwnica-Worms H, Roberts T M, McGlynn E
Research Department, Ciba-Geigy Limited, Basel, Switzerland.
Biochem J. 1992 Nov 1;287 ( Pt 3)(Pt 3):985-93. doi: 10.1042/bj2870985.
To obtain sufficient material for the biochemical and biophysical study of pp60c-src, we have utilized a recombinant pp60c-src baculovirus lacking the myristoylation site at codon 2. On infection of Sf9 cells, this virus produced large amounts of soluble non-myristoylated pp60c-src. The use of non-myristoylated pp60c-src (1) increases production of pp60c-src compared with the wild-type protein, (2) facilitates purification, (3) yields a stable product and (4) allows biochemical studies in the absence of detergents. Up to 20 mg of pp60c-src of greater than 95% purity has been purified from 6 litres of Sf9 cells grown in a bioreactor. One major and multiple minor forms of pp60c-src were separated by Mono Q f.p.l.c. Isoelectric focusing of purified pp60c-src species revealed heterogeneity, some of which could be attributed to differences in the tyrosine phosphorylation state of the enzyme. Kinetic analysis of non-myristoylated pp60c-src kinase in the presence of Mg2+ gave Km values for angiotensin II and ATP of 2 mM and 30 microM respectively and a Vmax. of 620 nmol/min per mg. The kinetic constants and metal ion preferences of a number of copolymers and peptide substrates have been compared. Polylysine and poly(GLAT), which was not phosphorylated by the pp60c-src kinase, dramatically activated autophosphorylation of Tyr-416, suggesting a conformation modulation of pp60c-src by charged polymers. This finding implies that Tyr-527 dephosphorylation is not sufficient for full activation of pp60c-src in vitro.
为了获得足够的材料用于pp60c-src的生化和生物物理研究，我们利用了一种重组的pp60c-src杆状病毒，该病毒在第2密码子处缺乏肉豆蔻酰化位点。感染Sf9细胞后，这种病毒产生了大量可溶性的非肉豆蔻酰化pp60c-src。使用非肉豆蔻酰化的pp60c-src：（1）与野生型蛋白相比，增加了pp60c-src的产量；（2）便于纯化；（3）产生稳定的产物；（4）允许在无去污剂的情况下进行生化研究。从生物反应器中生长的6升Sf9细胞中已纯化出高达20毫克、纯度大于95%的pp60c-src。通过Mono Q快速蛋白质液相色谱法分离出一种主要形式和多种次要形式的pp60c-src。对纯化的pp60c-src种类进行等电聚焦显示出异质性，其中一些可归因于该酶酪氨酸磷酸化状态的差异。在Mg2+存在下对非肉豆蔻酰化pp60c-src激酶的动力学分析得出，血管紧张素II和ATP的Km值分别为2 mM和30 microM，Vmax为每毫克620 nmol/min。比较了多种共聚物和肽底物的动力学常数和金属离子偏好性。未被pp60c-src激酶磷酸化的聚赖氨酸和聚（GLAT）显著激活了Tyr-416的自磷酸化，表明带电聚合物对pp60c-src的构象有调节作用。这一发现意味着在体外，Tyr-527去磷酸化不足以使pp60c-src完全激活。
