Rat Liver Alkaline Phosphatase

Hepatic alkaline phosphatase has been purified 42,000fold from rats whose bile ducts had been ligated for 24 hours, a procedure which increases hepatic alkaline phosphatase activity S-to IO-fold.The purified enzyme was obtained in 27% yield and was greater than 98% homogeneous as judged by polyacrylamide disc gel electrophoresis and meniscus depletion sedimentation equilibrium analysis.The molecular weight of the holoenzyme was 154,000.Rat liver alkaline phosphatase precipitated in 6 M guanidine-HCl but dissociated into two soluble subunits of identical molecular weight (71,200) in 6 M guanidine HCl plus 1% 2-mercaptoethanol.Polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate indicated a protein molecular weight of 155,000.The addition of 1% 2-mercaptoethanol caused the dissociation of alkaline phosphatase into two subunits of identical molecular weight, 75,000.The pH optimum for rat liver alkaline phosphatase was 10.2 with p-nitrophenylphosphate, 9.0 with phosphorylethanolamine and phosphorylcholine, 8.9 with AMP, ADP, and ATP, and 8.3 with pyrophosphate.Rat liver alkaline phosphatase appears to be a zinc-containing enzyme, loss of which results in irreversible inactivation.Magnesium is required for catalytic activity.Loss of activity after electrodialysis can be rapidly reversed by the addition of magnesium.Purified rat liver alkaline phosphatase has ATPase activity, but the V,,,*, of A'IP is only 12% that of p-nitrophenylphosphate, while its K,,, is similar, 0.8 mu and 1.0 ~IVI, respectively.The naturally occurring constituents of bile do not stimulate the ATPase activity of pure alkaline phosphatase.Pure rat liver alkaline phosphatase displayed no unusual affinity for phosphorylcholine, a substance suggested as the physiological substrate for this enzyme.