Multiple acid phosphatase in barley coleoptiles. Isolation and partial characterization of the 63 kDa soluble enzyme form

The soluble acid phosphatase (EC 3.1.3.2) from barley (Hordeum vulgare) coleoptiles was purified 46-fold by ammonium sulphate fractionation, DEAE-cellulose, Concanavalin A-Sepharose 4B and gel filtration chromatography. The molecular mass of the native enzyme was 63 kDa as determined by gel filtration on a Sephadex G-100 column. The subunit molecular mass of the purified enzyme, determined by SDS-PAGE, was 57.5 kDa. Of the numerous divalent cations tested, the purified enzyme was inhibited about 77% by 1 mM molybdate; Cu +2 , Mg +2 and Zn +2 also inhibited the enzyme, but at higher concentrations. The enzyme hydrolyzes a wide variety of natural and synthetic phosphate esters. In particular, this enzyme showed high specificity for inorganic pyrophosphate with a K m value of 0.19 mM. The pH dependence studies between pH 4.3-7.9 using p-nitrophenylphosphate (p-NPP) as substrate indicate the importance of two dissociable groups in the hydrolysis mechanism, one at pH 5.0 and the other at pH 5.8. The first probably represents the ionization of the substrate (p-NPP), while the second could be attributed to the dissociation of a histidine residue.