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130 kDa acid phosphatase from the liver of labeo rohita:isolation, purification and some kinetic properties.
Author(s)
Aisha Siddiqua Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan
Mehran Sherazi Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan
Rubina Naz Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan
Irshad Ali Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan
Ahmad Saeed Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan
Asma Saeed Department of Biological Sciences, Gomal University, Dera Ismail Khan, Pakistan
Abdul Haleem Shah Department of Biological Sciences, Gomal University, Dera Ismail Khan, Pakistan
Abdul Rahim Khan Department of Biological Sciences, Gomal University, Dera Ismail Khan, Pakistan
Mushtaq Ahmad Department of Biotechnology, University of Science and Technology, Bannu, Pakistan
Hidayatullah Khan Department of Biotechnology, University of Science and Technology, Bannu, Pakistan
Abstract
Summary: 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, CM-Cellulose and Sephacryl HR-200 columns. Nearly 500-folds purification was achieved. The molecular weight was estimated to be 120-130 kDa by polyacrylamide gel electrophoresis (PAGE) of native enzyme and 130 kDa by gel filtration on calibrated Sephadex G-100 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 50oC and temperature stability was 0o-50oC. 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 PO4 while pyridoxamine-5 PO4 showed poor inhibition. Metal ions such as Age, Cu2+, Zn2+ showed strong inhibition on the enzyme activity while other divalent ions like Mg2+, Mn2+ and Co2+ were found to be poor inhibitors. Modifiers like EDTA, methanol, ethanol, acetone and glycerol had no effect on the enzymes activity.
Publication Details
Page(s) 801-808
DOI DOI not available
Published Journal: Journal of Chemical Society of Pakistan, Volume: 31, Issue: 5, Year: 2009
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