乳牛胎盘硷性磷酸酶的提纯和性质

Studies on Purification of Alkaline Phosphatase from Cow placenta and Its Properties

通过正丁醇抽提、硫酸铵沉淀、DE-52离子交换层析和Con.A—Sepharose4B亲和层析,从乳牛胎盘中提取出硷性磷酸酶(ALP)。提纯倍数为109倍,比活为87金氏单位/mg蛋白。乳牛胎盘提取液经薄层聚丙烯酰胺等电聚焦电泳只出现1条酶带,pI值4.5;提取的ALP分子量为168000;对热稳定性差,于56℃下作用10min,其活性丧失84％;对磷酸苯二钠的km值为2.1mmol/L;L—苯丙氨酸和L—色氨酸对该酶有抑制作用,10mmol/L—苯丙氨酸使酶活性抑制27％,10mmol/L L—色氨酸使酶活性抑制49％;尿素对该酶的抑制作用随着尿素浓度的增加而逐渐加强,2mol/L尿素可使酶活性抑制87％;左旋咪唑对该酶有较强的抑制作用,10^(-4)mol/L左旋咪唑使酶活性抑制79％;该酶在pH<7的条件下不稳定,在pH7～11范围内较为稳定;最适pH为10.4;妊娠乳牛血清中不含有胎盘来源的ALP同工酶。

Alkaline phosphatase of cow placenta was purified by aprocedure involving extraction with butanol, ammonium sulfate fraction-ation and chromatography on DEAE-cellulose(DE-52) and concanavalinA-sepharose 4B. In electrophoresis, cow placental ALP formed a singleband of molecular weight 168000. Its isoelectric point was determined tobe 4. 5 by isoelectric focusing electrophoresis using a carrier ampholyte(pH3. 5- 10). The Km value for disodium phenyl orthophosphatemeasured in the standard assay system was 2. 1 mmol/L. It wasrelatively thermolabile and resulted in loss of activity by 84% byheating at 56℃ for ten minutes. The enzyme was strongly inhibited byL-levamisole, its activity decreased by 79% at 10^(-4)mol/L concentration.Urea inhibited the enzyme activity by 87% at 2mol/L. The results showedthat both L-phenylalanine and L-tryptophan slightly inhibited cow placentalALP. It was found that cow placental ALP isoenzyme was not presentin maternal serum during normal pregnancy.