PURIFICATION AND CHARACTERIZATION OF THE HUMAN PROTEIN-TYROSINE-PHOSPHATASE, PTP-MU, FROM A BACULOVIRUS EXPRESSION SYSTEM
PURIFICATION AND CHARACTERIZATION OF THE HUMAN PROTEIN-TYROSINE-PHOSPHATASE, PTP-MU, FROM A BACULOVIRUS EXPRESSION SYSTEM
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DOI：
10.1007/bf01076764
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发表时间：
1993-11-01
影响因子：
4.3
通讯作者：
TONKS, NK
中科院分区：
文献类型：
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作者：
BRADYKALNAY, SM;TONKS, NK
与PTPase类似的受体PTP mu在其细胞外片段上与细胞粘附分子的免疫球蛋白超家族成员具有结构相似性。在杆状病毒/Sf9细胞系统中表达了全长（200 kD）和仅表达细胞内PTPase结构域片段（80 kD）的构建体，并进行了纯化和鉴定。全长PTP以膜结合形式存在，而截断形式在可溶性部分被恢复。PTP比其他酪氨酸磷酸化的底物如髓鞘碱性蛋白（MBP）或合成肽EDNDYINASL更优先去磷酸化羧氨基甲基化和马来酰化的溶菌酶（RCML）衍生物。酶的可溶性，截断形式的酶的性质进行了详细的检查。pH最适值为7.5。K-m为400 nM， V-max为725 nmol/min/mg。这种形式的酶比全长PTP酶活性高2倍。全长型胰蛋白酶化抑制活性。钒酸盐和钼酸盐，有效的酪氨酸磷酸酶抑制剂，废除酶的活性。Zn++和Mn++离子、聚赖氨酸、聚葡聚糖/酪氨酸和精胺也有抑制作用。
The receptor like PTPase, PTP mu, displays structural similarity in its extracellular segment to members of the immunoglobulin superfamily of cell adhesion molecules. The full length form of PTP mu (200 kD) and a construct expressing only the intracellular PTPase domain-containing segment (80 kD) were expressed in the baculovirus/Sf9 cell system, purified and characterized. Full length PTP mu was membrane associated while the truncated form was recovered in the soluble fraction. PTP mu preferentially dephosphorylated a reduced carboxamidomethylated and maleylated derivative of lysozyme (RCML) over other tyrosine phosphorylated substrates such as myelin basic protein (MBP) or the synthetic peptide EDNDYINASL. The enzymatic properties of the soluble, truncated form of the enzyme were examined in detail. The pH optimum was 7.5. It dephosphorylated RCML with a K-m of 400 nM and a V-max of 725 nmol/min/mg. This form of the enzyme was 2 fold more active than full length PTP mu. Trypsinization of the full length form inhibited activity. Vanadate and molybdate, potent tyrosine phosphatase inhibitors, abolished activity of the enzyme. Zn++ and Mn++ ions, polylysine, poly-glu/tyr, and spermine were also inhibitory.