CHARACTERIZATION AND KINETIC-ANALYSIS OF THE INTRACELLULAR DOMAIN OF HUMAN-PROTEIN-TYROSINE-PHOSPHATASE-BETA (HPTP-BETA) USING SYNTHETIC PHOSPHOPEPTIDES
CHARACTERIZATION AND KINETIC-ANALYSIS OF THE INTRACELLULAR DOMAIN OF HUMAN-PROTEIN-TYROSINE-PHOSPHATASE-BETA (HPTP-BETA) USING SYNTHETIC PHOSPHOPEPTIDES
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DOI：
10.1042/bj2980395
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发表时间：
1994-03-01
影响因子：
4.1
通讯作者：
JIRIK, FR
中科院分区：
文献类型：
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作者：
HARDER, KW;OWEN, P;JIRIK, FR
在细菌中表达人蛋白酪氨酸磷酸酶β（HPTP β）（44 kDa）的胞内结构域，使用表位“标记”免疫亲和色谱法纯化，并在动力学曲线、底物特异性和酶活性的潜在调节剂方面进行表征。采用基于孔雀石绿色法的显色测定法检测HPTP β从磷酸肽释放的无机磷酸盐（P-i）。对该测定法进行修改以提高其灵敏度，使其适用于96孔微量滴定板格式，并提供50至1000 pmol之间的P-1线性检测。HPTP β的胞质结构域被钒酸盐、EDTA、肝素、聚（Glu，Tyr）（4：1）和锌离子强烈抑制。为了探索底物的偏好，这个PTODN，我们产生了13个残基的合成磷酸酪氨酸含有肽，对应于生理酪氨酸磷酸化的网站。使用四种不同的磷酸肽，HPTP β的k（cat）值在76和258 s（-1）之间。HPTP β的底物偏好顺序为src（Tyr-527）> PDGF-R（Tyr-740）> ERK 1（Tyr-204），远大于CSF-1 R（Tyr-708），K-m值范围为140 μ M至大于10 mM。亲和力的变化可能是由于所比较的四种磷酸肽之间的差异，特别是关于磷酸酪氨酸残基侧翼的带电氨基酸的特征。
The intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) (44 kDa) was expressed in bacteria, purified using epitope 'tagging' immunoaffinity chromatography, and characterized with respect to kinetic profile, substrate specificity and potential modulators of enzyme activity. A chromogenic assay based on the Malachite Green method was employed for the detection of inorganic phosphate (P-i) released from phosphopeptides by HPTP beta. This assay, modified so as to improve its sensitivity, was adapted to a 96-well microtitre plate format, and provided linear detection between 50 and 1000 pmol of P-i. The cytoplasmic domain of HPTP beta was strongly inhibited by vanadate, molybdate, heparin, poly(Glu, Tyr) (4:1) and zinc ions. In order to explore the substrate preferences of this PTPase, we generated 13-residue synthetic phosphotyrosine-containing peptides that corresponded to sites of physiological tyrosine phosphorylation. HPTP beta demonstrated k(cat) values between 76 and 258 s(-1) using four different phosphopeptides. The substrate preference of HPTP beta was in the order src(Tyr-527) > PDGF-R(Tyr-740) > ERK1(Tyr-204) much greater than CSF-1R(Tyr-708) with K-m values ranging from 140 mu M to greater than 10 mM. The variations in affinity were probably due to differences among the four phosphopeptides compared, particularly with respect to the character of the charged amino acids flanking the phosphotyrosine residue.