Title of Thesis
Isolation, Purification And Characterization Of
Zn++-Dependent Acid Phosphatase From Chicken’s Heart And
Its Comparison With The Enzyme Of Chicken’s Liver
Department Of Biological Sciences / Gomal
University, Dera Ismail Khan
|Number of Pages|
|Keywords (Extracted from title, table of contents and
abstract of thesis)|
Characterization, Zn++, Dependent Acid, Phosphatase,
Comparison, Enzyme, Liver, purified, ammonium, sulphate,
biochemical, catalysed, molecular
Low molecular weight
Zn++-dependent acid phosphatase from chicken ,s heart was partially
purified by ammonium sulphate precipitate,heat treatment at 60șC .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 65șC. 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
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 55șC.
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 ” mol
min-1 mg-1 at 37șC wheras the Km for 100 kDa enzyme from chicken ,s
liver was 0.8 mM and Vmax was12 ” 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
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.