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Isolation, Purification And Characterization Of Zn++-Dependent Acid Phosphatase From Chicken’s Heart And Its Comparison With The Enzyme Of Chicken’s Liver

SAEED, ASMA (2009) Isolation, Purification And Characterization Of Zn++-Dependent Acid Phosphatase From Chicken’s Heart And Its Comparison With The Enzyme Of Chicken’s Liver. PhD thesis, Gomal University, D.I. Khan.

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Abstract

Low molecular weight Zn++-dependent acid phosphatase from chicken ,s heart was partially purified by ammonium sulphate precipitate,heat treatment at 60ºC .CMCellulose chromatography and gel filtration on Sephadex G-100 to specific activity of 2.25 U/mg of protein at pH 6.0. A 100-fold purification was achieved with recovery of 8%. The enzyme had molecular weight 57 kDa as revealed by gel filtration and 29 kDa by SDS-polyacrylamide gel electrophoresis, indicating the dimeric nature of the enzyme.The enzyme showed maximum activity over a pH of 4.5- 6.0. The optimal temperature for the activity was 65ºC. Zn++-dependent acid phosphatase showed no p.nitrophenyl phosphate hydrolyzing activity in the absence of Zn++ions. The hydrolysis was observed in the presence of Zn++, Mn++and Co ++. Zinc showed maximal activation. Heavy metal ions such as Fe++, Cu++and Hg++ihibited the enzyme. Zn++-dependent acid phosphatase was inhibited by phosphate while EDTA, tartrate and fluoride had little or no effect on the activity which are the potent inhibitors of high molecular weight acid phosphatases. High molecular weight Zn++-dependent acid phosphatase was purified from chicken ,s liver with specific activity of 11 U/mg of protein and very small recovery. Purification achieved was 475-fold. The purified enzyme migrated as single band corresponding to 48 kDa on SDS-PAGE. The apparent molecular weight of the enzyme was estimated to be 110 kDa by gel filtration and consisted of two subunits. This enzyme was designated as 100 kDa in further study as we are not very much clear of its molecular weight because most of the enzymes from various sources have been reported in the range of 93-103 kDa. This discrepancy needs to be solved. Anyhow, the enzyme had optimal pH 5.5- 7.5 and optimal temperature of 55ºC. Comparative study of biochemical properties of 57 kDa and 100 kDa Zn++-dependent acid phosphatases was carried out. Km for 57 kDa Zn++-dependent acid phosphatase from chicken ,s heart on p.nitrophenyl phosphate was 0.6 mM at pH 6.0 with Vmax 2.5 µ mol min-1 mg-1 at 37ºC wheras the Km for 100 kDa enzyme from chicken ,s liver was 0.8 mM and Vmax was12 µ mol min-1 mg-1. 57 kDa enzyme significantly catalysed the hydrolysis of p.nitrophenyl phosphate, phenyl phosphate, phosphotyrosine, α- and β- naphthyl phosphate while 100 kDa enzyme caralysed the hydrolysis of these substrates to lesser rates. myo-inositol-1- phosphate and myo-inositol-2-phosphate were not hydrolysed by both enzymes in the presence of 5 mM Zn++ions at pH 6.0. 57 kDa enzyme catalysed the hydrolysis of myo-inositol-1-phosphate in the presence of 3 mM Mg++at pH 7.2 while 100 kDa enzyme did not hydrolyse myo-inositol-1-phosphate. myo-inositol-2-phosphate was also hydrolysed by 57 kDa enzyme but to lesser extent. This finding suggests that low molecular weight ( 57 kDa ) Zn++-dependent acid phosphatase possesses Mg++- dependent myo-inositol-1- phosphatase activity while high molecular weight ( 100 kDa ) Zn++-dependent acid phosphatase does not. Phosphate, tartrate, vanadate and molybdate were found strong inhibitors for both enzymes.These enzymes were inhibited competitively. 100 kDa enzyme was more sensitive to these inhibitors than 57 kDa enzyme. No other significant difference in biochemical properties between two enzymes from heart and liver was found.

Item Type:Thesis (PhD)
Uncontrolled Keywords:Isolation, Purification, Characterization, Zn++, Dependent Acid, Phosphatase, Chicken’s, Comparison, Enzyme, Liver, purified, ammonium, sulphate, biochemical, catalysed, molecular
Subjects:Biological & Medical Sciences (c) > Biological Sciences(c1)
ID Code:6298
Deposited By:Mr. Javed Memon
Deposited On:30 Jun 2011 14:29
Last Modified:30 Jun 2011 14:29

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