THE PURIFICATION AND CHARACTERIZATION OF A HUMAN DUAL-SPECIFIC PROTEIN-TYROSINE-PHOSPHATASE
THE PURIFICATION AND CHARACTERIZATION OF A HUMAN DUAL-SPECIFIC PROTEIN-TYROSINE-PHOSPHATASE
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DOI：
10.1074/jbc.270.8.3796
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发表时间：
1995-02-24
影响因子：
4.8
通讯作者：
DIXON, JE
中科院分区：
文献类型：
--
作者：
DENU, JM;ZHOU, GC;DIXON, JE
开发了一种表达和纯化方法，以获得适于动力学研究和结晶的量的重组人双特异性蛋白酪氨酸磷酸酶（PTH 4）VHR。均质重组蛋白的物理表征通过基质辅助激光解吸质谱法验证质量为20，500 +/- 100，证实了预期的NH 2-末端氨基酸序列，并证明蛋白质以单体形式存在。条件进行了开发，以获得适合于X射线结构测定的晶体。使用对应于MAP（177-189）（促分裂原活化蛋白）激酶（DHTGFLpTEpYVATR）的合成二磷酸化肽，设计了一种测定法，其允许测定二磷酸化肽在苏氨酸和酪氨酸残基上的去磷酸化的速率常数。二磷酸化肽比酪氨酸上的单磷酸化肽优选3-8倍。磷酸酪氨酸在DHTGFLpTEpYVATR上脱磷酸化的表观二级速率常数k（cat）/K-m为32，000 M（-1）s（-1），而磷酸苏氨酸的脱磷酸化为14 M（-1）s（-1）（pH 6）。DHTGFLpTEpYVATR与VHR的反应是有序的，首先发生酪氨酸的快速去磷酸化，然后是苏氨酸的缓慢去磷酸化。用F（NLe）（NLe）pTPpYVVTR获得了类似的结果，F（NLe）pTPpYVVTR是一种对应于MAP激酶样蛋白（JNK 1（180-189））的肽，其参与应激反应信号传导途径。
An expression and purification method was developed to obtain the recombinant human dual specific protein tyrosine phosphatase (PTPase) VHR in quantities suitable for both kinetic studies and crystallization. Physical characterization of the homogeneous recombinant protein verified the mass to be 20,500 +/- 100 by matrix-assisted laser desorption mass spectrometry, confirmed the anticipated NH2-terminal amino acid sequence and demonstrated that the protein exists as a monomer. Conditions were developed to obtain crystals which were suitable for x-ray structure determination. Using synthetic diphosphorylated peptides corresponding to MAP(177-189) (mitogen-activated protein) kinase (DHTGFLpTEpYVATR), an assay was devised which permitted the determination of the rate constants for dephosphorylation of the diphosphorylated peptide on threonine and tyrosine residues. The diphosphorylated peptides are preferred over the singly phosphorylated on tyrosine by 3-8-fold. The apparent second-order rate constant k(cat)/K-m for dephosphorlyation of phosphotyrosine on DHTGFLpTEpYVATR was 32,000 M(-1) s(-1) while dephosphorylation of phosphothreonine was 14 M(-1) s(-1) (pH 6). The reaction of DHTGFLpTEpYVATR with VHR is ordered, with rapid dephosphorylation on tyrosine occurring first followed by slow dephosphorylation on threonine. Similar results were obtained with F(NLe) (NLe)pTPpYVVTR, a peptide corresponding to a MAP kinase-like protein (JNK1(180-189)) which is involved in the stress response signaling pathway.