Action of Purified Milk Phosphatase on Phosphoserine and on Casein

The action of a purified alkaline phosphatase from milk on casein and phosphoserine has been studied. The milk phosphatase preparation has no proteolytic action on casein, but it splits inorganic phosphate from both casein and phosphoserine. The action of the milk phosphatase was studied at pH 8 to 9.5 with several concentrations of phosphoserine. The optimum action was at pH 9.5 or higher. Values for the kinetic constants KM and Vmax. were calculated from the data. The action of the milk phosphatase on phosphoserine was similar to its action on other simple phosphate esters. The milk phosphatase was considerably less reactive with casein (about 200 times more phosphatase was required for comparable release of phosphate), and the greatest activity was at pH 6 to 7. Reasons are given for not necessarily postulating a second enzyme for this activity, but rather that it might result from the complex nature and size of the casein molecule. Both calcium-sensitive α-casein and whole casein were dephosphorylated at comparable rates. The experiments were carried to 80% dephosphorylation of the casein, and presumably complete dephosphorylation could be accomplished with this phosphatase preparation. It is suggested that some dephosphorylation of casein might occur in milk and that special precautions may be required to obtain casein preparations of maximum and constant phosphorus content.