Alkaline phosphatase of human and calf small intestine. Purification and immunochemical characterization

Abstract

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.