Hiraga A, Munakata H, Hata K, Suzuki Y, Tsuiki S
Biochemistry Laboratory, Tohoku University, Japan.
Eur J Biochem. 1992 Oct 1;209(1):195-206. doi: 10.1111/j.1432-1033.1992.tb17277.x.
Utilizing three proteins plus tyrosine-glutamate copolymer as substrates, all of which are subjected to (near) stoichiometrical phosphorylation exclusively on tyrosine residues, we partially purified four different protein-tyrosine phosphatases (PTPases) from rat liver cytosol which differed in substrate preference. Of the four PTPases, tentatively termed L1, L2, L3, and L4, PTPase L1 was purified to apparent homogeneity by a procedure involving chromatography on DEAE-cellulose at pH 7.0, Blue Sepharose, DEAE-cellulose at pH 7.6, hydroxyapatite, Phenyl Sepharose, Mono Q, and TSKgel Heparin. PTPase L1 was purified about 7000-fold from the extract and 0.27 mg was isolated from 1000 g liver corresponding to a yield of 13% from the Blue Sepharose step where it had become freed from any other PTPases detectable by our assay procedure. The purified PTPase L1 showed a major protein band of 67 kDa on SDS/PAGE. Catalytically, PTPase L1 had a specific activity of about 6500 nmol Pi released min-1mg-1 toward tyrosine-glutamate copolymer phosphorylated on tyrosine residues. PTPase L1 exhibited very low sensitivities to PTPase inhibitors such as zinc acetate, sodium vanadate, and acidic compounds as compared with those of most of the PTPases purified thus far. Amino acid sequence analysis of the purified PTPase L1 revealed a partial peptide sequence showing similarity to the catalytic domain core sequences conserved in the PTPase family. PTPase L1 was most similar to a PTPase termed PTP1C encoded by a human breast carcinoma cDNA but the identity was 55% over 117 residues spanning nearly half of the catalytic domain of PTP1C. The analysis also revealed another partial peptide sequence (113 residues) 70% identical with the sequence corresponding to 68% of two adjacent copies of the src homology region 2(SH-2 domain) identified in PTP1C. Besides those peptide sequences, PTPase L1 had regional sequences which were 70-90% identical with the residues lying between the two SH-2 domains or between the more C-terminal SH-2 domain and the catalytic domain of the carcinoma PTPase.
利用三种蛋白质以及酪氨酸 - 谷氨酸共聚物作为底物，所有这些底物仅在酪氨酸残基上进行（近乎）化学计量的磷酸化，我们从大鼠肝脏胞质溶胶中部分纯化了四种不同的蛋白酪氨酸磷酸酶（PTP酶），它们在底物偏好上有所不同。在这四种PTP酶中，暂时命名为L1、L2、L3和L4，PTP酶L1通过以下步骤纯化至表观均一性：在pH 7.0的DEAE - 纤维素、蓝色琼脂糖凝胶、pH 7.6的DEAE - 纤维素、羟基磷灰石、苯基琼脂糖凝胶、Mono Q和TSKgel肝素柱上进行层析。PTP酶L1从提取物中纯化了约7000倍，从1000克肝脏中分离出0.27毫克，对应于蓝色琼脂糖凝胶步骤的产率为13%，在该步骤中它已从我们的检测方法可检测到的任何其他PTP酶中分离出来。纯化的PTP酶L1在SDS/PAGE上显示出一条67 kDa的主要蛋白带。在催化方面，PTP酶L1对酪氨酸残基磷酸化的酪氨酸 - 谷氨酸共聚物的比活性约为6500 nmol Pi释放·min⁻¹·mg⁻¹。与迄今为止纯化的大多数PTP酶相比，PTP酶L1对PTP酶抑制剂如醋酸锌、钒酸钠和酸性化合物表现出非常低的敏感性。纯化的PTP酶L1的氨基酸序列分析揭示了一个部分肽序列，该序列与PTP酶家族中保守的催化结构域核心序列具有相似性。PTP酶L1与一种由人乳腺癌cDNA编码的称为PTP1C的PTP酶最为相似，但在跨越PTP1C催化结构域近一半的117个残基上的同一性为55%。分析还揭示了另一个部分肽序列（113个残基），与PTP1C中鉴定的src同源区域2（SH - 2结构域）的两个相邻拷贝的68%对应的序列有70%的同一性。除了这些肽序列外，PTP酶L1还有一些区域序列，与癌PTP酶的两个SH - 2结构域之间或更靠近C末端的SH - 2结构域与催化结构域之间的残基有70 - 90%的同一性。
