Lau K H, Freeman T K, Baylink D J
J Biol Chem. 1987 Jan 25;262(3):1389-97.
An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.
一种具有磷酸酪氨酸蛋白磷酸酶活性的酸性磷酸酶已从牛皮质骨基质中纯化至表观均一。该酶活性的总产率大于25%，总纯化倍数约为2000倍，在pH 5.5和37℃下，比活性为每分钟每毫克蛋白质水解8.15 μmol对硝基苯磷酸酯。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（银染技术）上呈现单一蛋白条带，判断该纯化酶已被纯化。该酶可归类为5型抗酒石酸酸性磷酸酶同工酶。通过凝胶过滤法测定该酶活性的表观分子量为34,600，在还原剂存在下通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定为32,500，表明活性酶是单条多肽链。动力学评估显示，酸性磷酸酶活性似乎通过伪单底物双水解两步转移反应机制催化其反应，并受到磷酸根过渡态类似物的竞争性抑制。该酶活性对还原剂和几种二价金属离子也敏感。底物特异性评估表明，这种纯化的牛骨骼酸性磷酸酶能够水解核苷酸三磷酸和二磷酸、磷酸酪氨酸和磷酸酪氨酸组蛋白，但不能水解核苷酸单磷酸、磷酸丝氨酸、磷酸苏氨酸、磷酸丝氨酰组蛋白或低分子量磷酸酯。对磷酸酪氨酸蛋白磷酸酶活性的进一步检测表明，在固定底物浓度（50 nM磷酸组蛋白）下，该活性的最佳pH为7.0。对磷酸酪氨酸蛋白磷酸酶活性的动力学分析表明，纯化酶在pH 7.0时，从[32P]磷酸酪氨酸组蛋白水解[32P]磷酸根的表观Vmax约为每分钟每毫克蛋白质60 nmol，磷酸酪氨酸蛋白的表观Km约为450 nM磷酸基团。总之，这些研究结果代表了首次将骨骼酸性磷酸酶纯化至表观均一。我们观察到这种纯化的牛骨基质酸性磷酸酶能够在中性pH下使磷酸酪氨酸蛋白去磷酸化，这与我们提出的该酶可能在体内作为磷酸酪氨酸蛋白磷酸酶发挥作用的观点一致。
