Purification of alkaline phosphatase from bovine intestinal mucosa

Purification of alkaline phosphatase from bovine intestinal mucosa Crude APase (Boehringer, grade II) can be purified 

20-foId and completely recovered by the simple method of Landt et al. (1978) and Mossner et al. (1980). The enzyme is dialyzed against several changes of 10 mM Tris-HCl buffer, pH 8.0, over a 24 h period. The sample is applied to a column of L-histidyldiazo-benzylphosphonic acid-agarose (Sigma capacity about 100 U/ml), washed with Tris buffer and the enzyme is eluted with 10 mM 

Na2HP04. The enzyme is then dialyzed against the Tris buffer containing 0.1 mM ZnCl2and 1 mM MgCl2. The column is regenerated with Tris-HCl buffer. 

Under certain conditions the steady state has the form of the Michaelis-Menten equation (Section 9.1.2). Nevertheless, the equation for APase contains more factors than that of the simple reaction given in Section 9.1.2. For detailed kinetic considerations, Fernley (1971) and Reid and Wilson (1971) should be consulted. The simplified representation of Fig. 10.3 is also often complicated by other parameters e.g. only one active site per dimer seems to be active for the bacterial enzyme at low substrate coneentrations ( 10 M), whereas at higher concentrations both sites are active. Substrate activation, at high substrate concentrations ( 10 M), was noted by Heppel et al. (1962) but not at high ionic strength (Simpson and Vallee, 1970). 

Fernley (1971) discussed the influence of different buffers on bovine 

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