Purification and characterization of an acid phosphatase from peanut (Arachis hypogaea) seed

Abstract

An acid phosphatase (EC 3.1.3.2) from peanut (Arachis hypogaea L.) seed has been purified 433-fold by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. The purified preparation was found to be homogeneous by electrophoresis and gel filtration. The molecular weight of the enzyme was estimated to be approximately 240 000 and it was found to be composed of six identical subunits, each with an apparent molecular weight of 42 500. Following isoelectric focusing, the isolectric point (pI) of the enzyme was found to be around pH 5.6. The apparent Km value with p-nitrophenyl phosphate as substrate was 2 ? 10−1 μM. The enzyme was inhibited by Hg2+, Fe2+, Cu2+, Zn2+, Pb2+, and F−. Higher concentrations (2–50 μM) and long incubation periods (60–90 min) with Ca2+ and Mg2+ ions were shown to activate the enzyme. This enzyme showed no effect toward phosphorylated sugars but appear to hydrolyze ATP, ADP, AMP, and β-glycerophosphate.