Rat alkaline phosphatase. I. Purification and characterization of the enzyme from osteosarcoma: generation of monoclonal and polyclonal antibodies.
Rat alkaline phosphatase. I. Purification and characterization of the enzyme from osteosarcoma: generation of monoclonal and polyclonal antibodies.
复制标题
DOI：
10.1016/0003-9861(87)90076-2
复制
发表时间：
1987-04
影响因子：
3.9
通讯作者：
B. Nair;R. Majeska;G. Rodan
中科院分区：
文献类型：
--
作者：
B. Nair;R. Majeska;G. Rodan
用丙酮沉淀法从大鼠骨肉瘤中提取碱性磷酸酶（AP），经DEAE-纤维素、Sephacryl S-200和羟基磷灰石柱层析，得到纯度大于90%的AP。纯化的酶在最适pH（10.5）下的比活力为759单位/ mg蛋白，对磷酸对硝基苯酯的Km为0.8 mm。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上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是骨-肝-肾AP同工酶的特征。单克隆和多克隆抗AP抗体的制备和表征。多克隆兔抗血清定量沉淀纯化AP制剂和组织提取物的活性，但不抑制AP催化活性。当用125 I-AP进行竞争试验时，这种抗血清对热灭活酶的活性几乎低10倍。两种不同的单克隆抗体在单独测试时各自在免疫沉淀AP中部分有效;然而，它们一起沉淀了超过90%的AP活性。
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 aKmof 0.8 mmforp-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 using125I-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.