Wang S, Lehr R, Stevis P, Wang X M, Trevillyan J, Faltynek C R
Department of Immunology, Sterling Winthrop, Inc., Collegeville, Pennsylvania 19426.
Arch Biochem Biophys. 1994 Nov 15;315(1):60-7. doi: 10.1006/abbi.1994.1471.
p56lck, which is a lymphoid-restricted tyrosine kinase, plays a significant role both in activation of mature T lymphocytes and in T cell development. Recombinant human p56lck was expressed in a baculovirus system and purified to > 95% purity. Purified recombinant p56lck was found to have enzymatic characteristics indistinguishable from those of the native enzyme expressed in the Jurkat human T lymphocytic cell line. The comparisons of recombinant and native p56lck were performed using an assay in which the enzyme was immobilized with antibodies onto the wells of a microtiter plate, enabling in situ purification of the native enzyme. Further characterization of the purified recombinant p56lck revealed significantly different enzyme concentration curves for peptide phosphorylation by immobilized and soluble p56lck. In particular, there was very low activity at low concentrations of enzyme assayed in solution. The activity at low enzyme concentrations could be substantially increased by preincubation of high concentrations of enzyme with ATP prior to dilution for the peptide phosphorylation reaction. We examined the relationship between autophosphorylation and activation for peptide phosphorylation. As for peptide phosphorylation, nonlinear enzyme concentration curves were also observed for autophosphorylation in solution, with very low levels of autophosphorylation at low enzyme concentrations. There was a correlation between the enzyme concentration dependency for autophosphorylation and for preincubation with ATP for maximal enhancement of subsequent peptide phosphorylation. The results suggest that autophosphorylation activates p56lck for phosphorylation of exogenous substrates and that high enzyme concentrations accelerate intermolecular autophosphorylation and enzyme activation.
p56lck是一种淋巴细胞特异性酪氨酸激酶，在成熟T淋巴细胞的激活以及T细胞发育过程中均发挥着重要作用。重组人p56lck在杆状病毒系统中表达，并纯化至纯度大于95%。经发现，纯化后的重组p56lck具有与Jurkat人T淋巴细胞系中表达的天然酶无法区分的酶学特性。重组p56lck与天然p56lck的比较是通过一种检测方法进行的，在该方法中，酶通过抗体固定在微量滴定板的孔中，从而实现天然酶的原位纯化。对纯化后的重组p56lck的进一步表征显示，固定化p56lck和可溶性p56lck进行肽磷酸化反应时，酶浓度曲线存在显著差异。特别是，在溶液中检测低浓度酶时活性非常低。在进行肽磷酸化反应稀释之前，将高浓度酶与ATP预孵育，可显著提高低酶浓度时的活性。我们研究了自身磷酸化与肽磷酸化激活之间的关系。对于肽磷酸化，在溶液中自身磷酸化也观察到非线性酶浓度曲线，低酶浓度时自身磷酸化水平非常低。自身磷酸化的酶浓度依赖性与为最大程度增强后续肽磷酸化而与ATP预孵育之间存在相关性。结果表明，自身磷酸化激活p56lck以进行外源底物的磷酸化，并且高酶浓度加速分子间自身磷酸化和酶激活。
