ImmobilizedE. coli Alkaline Phosphatase

We have immobilized E.coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B. This preparation has several advantages over the soluble enzyme. The immobilized enzyme is easily separable from other constituents in incubation mixtures. The immobilized enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg²⁺. The insoluble and soluble phosphatases removed 75 and77%, respectively, of the inorganic phosphorus from casein. The immobilized enzyme inactivated two enzymes believed to be active in the phosphorylated state, acyl-CoA : cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by 89%. The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or membrane-bound enzymes and proteins is discussed.