Human liver acid phosphatases: purification and properties of a low-molecular-weight isoenzyme.
Human liver acid phosphatases: purification and properties of a low-molecular-weight isoenzyme.
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人肝酸性磷酸酶：低分子量同工酶的纯化和特性。
DOI：
10.1016/0003-9861(82)90054-6
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发表时间：
1982
影响因子：
3.9
通讯作者：
VanEtten,RL
中科院分区：
文献类型：
--
作者：
Taga,EM;VanEtten,RL
通过硫酸铵分级、酸处理和带有离子亲和洗脱的 SP-Sephadex 离子交换色谱法，将低分子量人肝酸性磷酸酶纯化 2580 倍至同质。纯化的酶含有单条多肽链，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定分子量为14,400。报告了该酶 (E) 的氨基酸组成。使用磷酸对硝基苯酯作为底物 (S) 的 pH 依赖性研究揭示了底物电离 (pKa5.2) 的影响以及 pKa 值为 7.8 的 ES 复合物中基团的参与。该酶很容易被重金属离子等巯基试剂失活。用碘乙酸和碘乙酰胺对酶进行烷基化导致酶完全失活，并且磷酸根离子的存在可以防止这种失活。该酶也可通过焦碳酸二乙酯处理而失活；磷酸根离子提供针对该试剂的保护。该酶的底物特异性对于酸性磷酸酶来说是不寻常的。在测试的许多烷基和芳基磷酸单酯中，唯一可能被该酶水解的生理底物是黄素单核苷酸，它的 V 在 pH 5.0 时比磷酸对硝基苯酯大 3 倍，在 pH 7.0 时大 6 倍。然而，该酶还在 pH 5.0 时催化乙酰磷酸水解，其速度比报道的人红细胞酰基磷酸酶大八倍。
A low-molecular-weight human liver acid phosphatase was purified 2580-fold to homogenity by a procedure involving ammonium sulfate fractionation, acid treatment, and SP-Sephadex ion-exchange chromatography with ion-affinity elution. The purified enzyme contains a single polypeptide chain and has a molecular weight of 14,400 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of this enzyme (E) is reported. A pH dependence study usingp-nitrophenyl phosphate as a substrate (S) revealed the effect of substrate ionization (pKa5.2) and the participation of a group in the ES complex having a pKavalue of 7.8. The enzyme is readily inactivated by sulfhydryl reagents such as heavy metal ions. Alkylation of the enzyme with iodoacetic acid and iodoacetamide causes complete inactivation of the enzyme and this inactivation is prevented by the presence of phosphate ion. The enzyme is also inactivated by treatment with diethyl pyrocarbonate; protection against this reagent is afforded by phosphate ion. The substrate specificity of this enzyme is unusual for an acid phosphatase. Of the many alkyl and aryl phosphomonoesters tested, the only possibly physiological substrate hydrolyzed by this enzyme was flavin mononucleotide, which exhibits a V which is 3-fold larger at pH 5.0 and 6-fold larger at pH 7.0 than that forp-nitrophenyl phosphate. However, the enzyme also catalyzes the hydrolysis of acetyl phosphate at pH 5.0 with a velocity eight times larger than that reported for an acyl phosphatase from human erythrocytes.