Biochem/Physiol Actions | Alkaline phosphatase dephosphorylates proteins and nucleic acids. Alkaline phosphatase is a dimer consisting of two equal subunits.The enzyme is a glycoprotein containing approximately 12% carbohydrate (6% hexoses and 6% other neutral sugars). Each molecule of alkaline phosphatase contains four zinc atoms and four disulfide bridges. Maximal activity with alkaline phosphatase is achieved in the presence of magnesium. It catalyzes the hydrolysis of phosphate monoesters such as p-nitrophenyl phosphate, phenyl phosphate, phenolphthalein phosphate, α-glycerol phosphate, β-glycerol phosphate, 2-phosphorylglycerate, triosephosphate, glucose-6-phosphate, glucose 1-phosphate, fructose 1-phosphate, fructose 6-phosphate, adenosine 5-phosphate adenosine 3-phosphate, phosphoenolpyruvate, and β-nicotinamide adenine dinucleotide phosphate. Arsenate, cysteine, iodine, inorganic phosphate, pyrophosphate, diisopropyl phosphate, triphenylphosphate, diisopropyl fluorophosphate, and L-phenylalanine are some of the strong inhibitors of alkaline phosphatase. The enzyme is most stable in the pH range 7.5-9.5.2. The pH optimum for enzymatic activity is pH 8-10. |
Application | Alkaline phosphatase is used for conjugation to antibodies and other proteins for ELISA, Western blotting, and histochemical detection. It is routinely used to dephosphorylate proteins and nucleic acids. It may be used for protein labeling when high sensitivity is required. Alkaline phosphatase may be also be used to dephosphorylate the 5′-termini of DNA or RNA to prevent self-ligation. DNA or RNA can also be tagged with radiolabeled phosphate (via T4 polynucleotide kinase) after dephosphorylation with alkaline phosphatase. Product P8361 has been used to dephosphorylate Dishevelled (Dvl) , which is a key component in the Wnt/β-catenin signaling pathway. An assay, using this ALP enzyme, was developed to assess the cryoprotective activity of two proteins CRP-1 and CRP-2. The enzyme has been used for inhibitory studies of cyclic nucleotide analogs that inhibit prostatic acid phosphatase using a fluorogenic assay. It is commonly used as a "reporter" in detection systems, in which it is conjugated to a protein (antibody, streptavidin, etc.) that specifically recognizes a target molecule. Alkaline phosphatase has also been used to dephosphorylate casein. |
