Khattab M, Pfleiderer G
Hoppe Seylers Z Physiol Chem. 1976 Mar;357(3):377-91. doi: 10.1515/bchm2.1976.357.1.377.
Alkaline phosphatase from human and calf small intestines has been prepared and purified until homogeneous, as judged by polyacrylamide gel electrophoresis, by means of the following techniques: n-butanol extraction, ammonium sulfate precipitation, acetone fractionation, ion exchange chromatography and isoelectric focusing in a sucrose density gradient. Three and two alkaline phosphatase froms from human and calf small intestines, respectively could be isolated by preparative isolectric focusing. The relative amounts of these components are not constant, but they have the same catalytic properties, suggesting that they may embody a common protein core with an identical active centre(s). Precipitating antisera for alkaline phosphatase from human and calf intestine have been prepard in rabbits by intramuscular, dermal, subcutaneous and intravenous administration of the pure major component of each enzyme species. Both antisera precipitate completely their homologous as well as their heterologous antigens (intestinal enzyme) and showed partial identity with placental alkaline phosphatase. There was no reaction with alkaline phosphatase from bone, liver heart, spleen, lung, stomach, pancreas, brain, bile-gall bladder and erythrocytes. Alkaline phosphatase preparation from human kidney contains a minor component of the intestinal type, beside many multiple forms which, on treatment with neuramindase, became identical in their electrophoretic and biochemical properties. At least eight multiple forms of the placental enzyme could be shown by isoelectric focusing in polyacrylamide gel. On treatment with neuraminidase these forms became less charged and only four forms remained. All forms were immunologically identical using either anti placental-enzyme or anti intestinal-AP serum. Monospecific antisera against human or calf intestinal alkaline phosphatase were obtained by absorption with purified placental enzyme. This monospecific anti intestinal-AP serum could be used for an immunological quantitative determination of the intestinal isoenzyme in sera or other liquids in the presence of other alkaline phosphatase isoenzymes.
已通过以下技术制备并纯化了来自人及小牛小肠的碱性磷酸酶，直至通过聚丙烯酰胺凝胶电泳判断其达到均一状态：正丁醇萃取、硫酸铵沉淀、丙酮分级分离、离子交换色谱以及在蔗糖密度梯度中进行等电聚焦。通过制备性等电聚焦分别可从人及小牛小肠中分离出三种和两种碱性磷酸酶形式。这些组分的相对含量并不恒定，但它们具有相同的催化特性，这表明它们可能体现了具有相同活性中心的共同蛋白质核心。通过肌肉内、皮肤、皮下和静脉内给予每种酶的纯主要成分，已在兔中制备了针对人及小牛肠碱性磷酸酶的沉淀抗血清。两种抗血清均可完全沉淀其同源以及异源抗原（肠酶），并与胎盘碱性磷酸酶显示部分同一性。与来自骨、肝、心、脾、肺、胃、胰腺、脑、胆囊和红细胞的碱性磷酸酶无反应。来自人肾的碱性磷酸酶制剂除了许多多种形式外，还含有一种肠道型的次要成分，用神经氨酸酶处理后，这些多种形式在电泳和生化特性上变得相同。通过聚丙烯酰胺凝胶等电聚焦可显示胎盘酶至少有八种多种形式。用神经氨酸酶处理后，这些形式的电荷减少，仅剩下四种形式。使用抗胎盘酶血清或抗肠碱性磷酸酶血清，所有形式在免疫学上都是相同 的。通过用纯化的胎盘酶吸收获得了针对人或小牛肠碱性磷酸酶的单特异性抗血清。这种单特异性抗肠碱性磷酸酶血清可用于在存在其他碱性磷酸酶同工酶的情况下对血清或其他液体中的肠同工酶进行免疫定量测定。
