Alkaline phosphatase APase

APases (orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1) are found primarily in animal tissues and microorganisms. APases used in EIA are isolated from bovine intestinal mucosa or from E. coli. These enzymes have considerable differences in their properties and should not, as often done, be assayed under identical conditions. 

APases hydrolyze numerous phosphate esters, such as those of primary and secondary alcohols, phenols and amines (Levine, 1974). One unit of activity of APase corresponds to the hydrolysis of 1.0 pmole of p-nitrophenyl phosphate (p-NPP) per min (in 100 mM glycine, 1 mM ZnCb, 1 mM MgCl2 and 6 mM p-NPP, pH 10.4 or in 1 M diethanolamine, 0.5 mM MgCU and 15 mM p-NPP, pH 9.8). The bovine enzyme generally has a specific activity of 1000 and 2000 U/mg in these two buffers, respectively, at 37°C. At 25°C, activity is reduced to about half. This demonstrates that buffers may have a marked influence on the enzymatic activity of APases which explains the great differences in activity given for commercial preparations. Assays with p-NPP above 30°C suffer from the spontaneous hydrolysis of this substrate, with serious consequences for the enzyme kinetics (see below). The bacterial enzyme has lower activity than the bovine intestinal enzyme. 

A major reason for the popularity of APase for EIA is its absence from higher plants. However, this enzyme is particularly abundant in animal and human tissues involved in nutrient transport (hence its isolation from intestinal mucosa), and in developing tissues and secretory organs, but it is not found in significant amounts in muscle. 

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Alkaline phosphatases (APases) are nonspecific phosphomonoesterases with optimal activities at alkaline pH ( 9), as opposed to acid phosphatases which are optimally active at low pH ( 5). APases catalyze the hydrolysis of a wide variety of phosphomonoesters (ROP) to the corresponding alcohols (ROH) and inorganic phosphates (Pj) (Scheme 5.1). 

While having three metal ions in an enzyme active site is uncommon, it is not unique to PLCBc. The well-known alkaline phosphatase from E. coli (APase) contains two zinc ions and a magnesium ion [67], whereas the a-toxin from Clostridiumperfringens [68]. and the PI nuclease from Penicillium citrinum [69] each contain three zinc ions. Indeed, the zinc ions and coordinating ligands of PI nuclease bear an uncanny resemblance to those of PLCBc as the only differ-... 

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

This section focuses on two most widely used APases, a bacterial alkaline phosphatase (BAP) from E. coli and a mammalian APase from calf intestinal mucosa (CIAP), to illustrate various strategies common to the application of APases. For the sake of general information on the diversity of mammalian APases, a brief description is given for human APases. 

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