Purification and characterization of a low-molecular-weight acid phosphatase--a phosphotyrosyl-protein phosphatase from bovine heart.
Purification and characterization of a low-molecular-weight acid phosphatase--a phosphotyrosyl-protein phosphatase from bovine heart.
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DOI：
10.1016/0003-9861(90)90084-c
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发表时间：
1990-10
影响因子：
3.9
通讯作者：
Z. Zhang;R. L. Etten
中科院分区：
文献类型：
--
作者：
Z. Zhang;R. L. Etten
低分子量的酸性磷酸酶，这是最近被证明是磷酸酪氨酰蛋白磷酸酶的一组代表纯化到均匀的牛心。该酶是一种分子量为18 kDa的单体，等电点为7.0。在280 nm处，吸收系数E1cm1%为9.65。该酶对底物磷酸硝基苯酯和酪氨酸磷酸的最适pH分别为5.3和6.0。当在pH 5和37 °C下测量时，该酶对对硝基苯基磷酸和酪氨酸O-磷酸的比活性分别为114和86 μmol min-1 mg-1，而Km值分别为0.38和14 mm。该酶对芳基单磷酸酯具有高度特异性，对脂肪族磷酸酯几乎没有或没有活性，除了黄素单胞苷肽（FMN）及其某些结构类似物之外。如31 P NMR数据所示，对FMN的活性是由于（商业）样品中存在的八种组分之一的水解。酒石酸和钒酸盐都是强抑制剂，抑制常数分别为37和29 μm;酒石酸和氟化物对酶活性影响不大。盐酸胍对酶的可逆变性有两个阶段，其中间点分别为0.25和1.75m。氨基酸组成与其他组织中的低分子量酸性磷酸酶同源。该酶对低分子量人肝酸性磷酸酶具有免疫交叉反应性。单体蛋白上有7 ~ 8个可接近的半胱氨酸，其中至少有一个是酶活性所必需的。该酶还具有磷酸转移酶活性，例如将磷酸从-硝基苯磷酸转移到多种醇受体。
A low-molecular-weight acid phosphatase that is representative of a group recently shown to be phosphotyrosyl protein phosphatases was purified to homogeneity from bovine heart. The enzyme was a monomer with a molecular mass of 18 kDa and had an isoelectric point of 7.0. The absorption coefficient,E1cm1%was 9.65 at 280 nm. The enzyme had pH optima of 5.3 and 6.0 with the substratesp-nitrophenyl phosphate and tyrosine phosphate, respectively. When measured at pH 5 and 37 °C, the enzyme had specific activities of 114 and 86 μmol min−1mg−1forp-nitrophenyl phosphate and tyrosineO-phosphate, respectively, while theKmvalues were 0.38 and 14 mm. The enzyme was highly specific for aryl monophosphate esters and showed little or no activity toward aliphatic phosphate esters, with the remarkable exception of flavin mononucleotide (FMN) and certain of its structural analogs. As shown by31P NMR data, the activity toward FMN was due to the hydrolysis of one of the eight components present in the (commercial) sample. Both molybdate and vanadate were potent inhibitors, with inhibition constants of 37 and 29 μm, respectively; tartrate and fluoride had little effect on enzymatic activity. A two-stage reversible denaturation of the enzyme by guanidine HCl was observed with midpoints of 0.25 and 1.75m, respectively. The amino acid composition was homologous to the lowmolecular-weight acid phosphatases from other tissue. The enzyme showed immunological cross-reactivity against low-molecular-weight human liver acid phosphatase. There were 7 or 8 accessible cysteines on the monomeric protein and at least one was essential for enzyme activity. The enzyme also had phosphotransferase activity, for example transferring phosphate fromp-nitrophenyl phosphate to a wide variety of alcohol acceptors.