Abstract
Pyrophosphatase (PPase, EC 3.6.1.1) responsible to the hydrolysis of pyrophosphate (PPi) in muscle was purified from bighead carp ( Aristichthys nobilis), and characterized in detail herein for the first time. PPase was extracted with 20 mmol/L Tris-HCl buffer (pH 7.0) containing 0.05 mol/L KCl, followed by heat treatment and ammonium sulfate precipitation. Then it was purified by deithylaminoethyl-cellulose (DE52) and Sephacryl S-300 chromatography, subsequently resulted in a 114.5-fold purification. The molecular mass of the PPase was defined to be 50 kDa with two subunits. The optimum pH of the purified PPase was around 8.0, and its optimum temperature was confirmed to be 50 °C. Magnesium ion was necessary to the activity of PPase. An excess of PPi over Mg 2+ resulted in inhibition of PPase. When the Mg 2+/PPi molar ratio was 1:1, the maximal enzyme activity was obtained. In addition, PPase converted PPi to orthophosphate (Pi) stoichiometrically with a K m of 1.98 mmol/L under the condition of 5 mmol/L Mg 2+. Ethylene diamine tetraacetate (EDTA) activated the activity of PPase at low concentration, however, it consumingly did inhibit the enzyme activity at high concentration.
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