High molecular weight acid phosphatase of chicken liver: Separation and characterization

High molecular weight acid phosphatase was purified from chicken liver with a specific activity of 21 U/ mg and a recovery of 1 %. Purification was achieved 550 fold. The enzyme was seemed to be homogeneous on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was estimated to be 100 killo daltons (kDa) by gel filtration on Sephadex G-100 and 50 kDa by SDS-PAGE. K_m against para-nitrophenyl phosphate and phenyl phosphate at pH 5.5 were found to be 0.11mM and 0.15mM respectively, while V_max for these two substrates were 22.9 Umin^-1mg ^-1 and 19.7 Umin^-1mg^-1 of protein respectively. The enzyme was strongly inhibited by fluoride, tartrate, phosphate, vanadate and molybdate and were found competitive inhibitors. Para-nitrophenyl phosphate, phenyl phosphate and α-naphthyl phosphate were found good substrates. Other substrates were also hydrolyzed but at slower rates. No activation was observed with purine basis, glycerol or methanol etc. and thus not possessing phosphotransferase activity..