There are a multitude of enzymes that hydrolyze ATP or GTP to form ADP or GDP and inorganic phosphate (Pi). The Enzyme Linked Inorganic Phosphate Assay (ELIPA) from Cytoskeleton Inc. allows one to measure the phosphate released during hydrolysis on a real time basis. Thus a kinetic assay technique is produced for your enzyme’s activity. Particular purified enzymes that are applicable to this analysis are signaling phosphatases, apyrase, kinesin motors (see Fig. 1), metabolic enzymes, membrane transporters and alkaline phosphatase. Generally this assay is useful for enzymes with Kcat above 0.1, if your enzyme or the family of enzymes has a lower Kcat then the CytoPhosTM Phosphate Assay (Cat. # BK054) is useful. If your enzyme has a very low activity i.e. small G-proteins, the non-radioactive GAP assay (Cat. # BK105) or the radioactive alternative, EasyRad Phosphate Assay (Cat. # BK055) is ideal.
The assay is an adaptation of a method originally described by Webb for the measurement of glycerol kinase plus D-glyceraldehyde ATPase activity and for actin activated myosin ATPase (1). The assay is based upon an absorbance shift (330-360 nm) that occurs when 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) is catalytically converted to 2-amino-6-mercapto-7-methyl purine in the presence of inorganic phosphate (Pi). The reaction is catalyzed by purine nucleoside phosphorylase (PNP). One molecule of inorganic phosphate will yield one molecule of 2-amino-6-mercapto-7-methyl purine in an essentially irreversible reaction (2). Thus, the absorbance at 360 nm is directly proportional to the amount of Pi generated in the reaction.
