Nair B C, Majeska R J, Rodan G A
Arch Biochem Biophys. 1987 Apr;254(1):18-27. doi: 10.1016/0003-9861(87)90076-2.
Alkaline phosphatase (AP) was purified to over 90% homogeneity from rat osteosarcoma by acetone precipitation followed by chromatography on DEAE-cellulose, Sephacryl S-200, and hydroxyapatite. The purified enzyme had a specific activity of 759 units/mg protein at its optimal pH (10.5), and a Km of 0.8 mM for p-nitrophenylphosphate. The enzyme's apparent subunit molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82,000 Da. The heat-inactivation profile and homoarginine inhibition were characteristic of the bone-liver-kidney AP isoenzyme. Monoclonal and polyclonal anti-AP antibodies were prepared and characterized. Polyclonal rabbit antiserum quantitatively precipitated the activity from purified AP preparations and tissue extracts but did not inhibit AP catalytic activity. This antiserum was almost 10-fold less active against heat-inactivated enzyme when tested in a competition assay using 125I-AP. Two distinct monoclonal antibodies were each partly effective in immunoprecipitating AP when tested individually; however, together they precipitated over 90% of the AP activity.
通过丙酮沉淀，然后在DEAE - 纤维素、Sephacryl S - 200和羟基磷灰石上进行层析，从大鼠骨肉瘤中纯化出碱性磷酸酶（AP），纯度超过90%。纯化后的酶在其最适pH（10.5）下的比活性为759单位/毫克蛋白质，对磷酸对硝基苯酯的Km值为0.8 mM。在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上，该酶的表观亚基分子量为82,000 Da。热失活曲线和高精氨酸抑制作用是骨 - 肝 - 肾AP同工酶的特征。制备并鉴定了单克隆和多克隆抗AP抗体。多克隆兔抗血清能从纯化的AP制剂和组织提取物中定量沉淀活性，但不抑制AP催化活性。在使用125I - AP的竞争试验中测试时，该抗血清对热失活酶的活性几乎低10倍。两种不同的单克隆抗体单独测试时，各自在免疫沉淀AP方面部分有效；然而，它们一起能沉淀超过90%的AP活性。
