Purification and characterization of four different alkaline phosphatases from Spirographis spallanzanii

Alkaline phosphatases (APs) present in Spirographis spallanzanii were extracted by water homogenization of butanol treatment. The enzymes solubilized as above were purified by acetone fractionation and then by DEAE-cellulose and Sephadex G-200 chromatography; separation of four AP forms was achieved, which were characterized by studying their molecular and catalytic properties. The various APs differ in molecular weight, electrophoretic mobility, optimum pH, but show similar Km values and substrate inhibition pattern. Kinetic studies carried out with several inhibitors show, in particular, the existence in these APs of a second binding site and suggest a possible role for them in the metabolism of phosphoric esters of the sugars.