130 kDa Acid Phosphatase from the Liver of Labeo Rohita: Isolation, Purification and some Kinetic Properties

Abstract

An isoenzyme of high molecular weight acid phosphatase (HM-ACP) from the liver of fish Rohu (Labeo Rohita) was isolated and purified to homogeneity. The enzyme had specific activity of 14.96 U/mg and a recovery of about 4 %. The purification procedure included ammonium sulphate precipitation and series of chromatographic separations on SP-Sephadex C-50, CMCellulose and Sephacryl HR-200 columns. Nearly 500-folds purification was achieved. The molecular weight was estimated to be 120-130 kDa by polyacfylamide gel electrophoresis (PAGE) of native enzyme and 130 kDa by gel filtration on calibrated Sephadex G-IOO column. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced & non - reduced conditions showed a band corresponding to 66 kDa confirming the dimeric nature of enzyme. Para nitrophenyl phosphate and flavin mononucleotide were hydrolyzed effectively by the enzyme and found to be good substrates. Optimum temperature for the enzyme was 50t and temperature 00 stability was 0-50C. Similarly optimum pH for the enzyme was 5.4 and pH stability was 4.8-6.0. The Km for the p-nitrophenyl phosphate was estimated to be 0.15 mM. The enzyme was competitively inhibited by the phosphate, vanadate, molybdate, tartrate, fluoride and pyridoxal-5/PO* while pyridoxarnine-5/-P04 showed poor inhibition. Metal ions such as Ag+, Cut Zn++ showed strong inhibition on the enzyme activity while other divalent ions like Mg+, and Co" were found to be poor inhibitors. Modifiers like EDTA, methanol, ethanol, acetone and glycerol had no effect on the enzymes activity.