Alkaline Phosphatase of Human and Calf Small Intestine. Purification and Immunochemical Characterization

Abstract

Alkaline phosphatase [EC 3.1.3.1, AP] from human and calf small intestines was prepared and purified until homogenous, 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 2 AP forms from human and calf small intestines, respectively, could be isolated by preparative isoelectric 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 center(s). Precipitating antisera for AP from human and calf intestine were prepared in rabbits by i.m., dermal, s.c. and i.v. administration of the pure major component of each enzyme species. Both antisera precipitate completely their homologous and heterologous antigens (intestinal enzyme) and showed partial identity with placental AP. There was no reaction with AP from bone, liver, heart, spleen, lung, stomach, pancreas, brain, bile-gall bladder and erythrocytes. AP preparation from human kidney contains a minor component of the intestinal type, beside many multiple forms which, on treatment with neuraminidase [EC 3.2.1.18], became identical in their electrophoretic and biochemical properties. At least 8 multiple forms of the placental enzyme were shown by isoelectric focusing in polyacrylamide gel. On treatment with neuraminidase these forms became less charged, and only 4 forms remained. All forms were immunologically identical using either anti placental-enzyme or anti-intestinal-AP serum. Monospecific antisera against human or calf intestinal AP 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 AP isoenzymes.