Isolation and characterization of the extracellular acid phosphatase of Leishmania Donovani promastigotes
Date
1992
Authors
Li, Fengchun
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Abstract
An extracellular acid phosphatase was purified from the culture supernatants of L. donovani promastigotes by using ultrafiltration, Con-A and lentil lectin affinity chromatography, Mono-Q ion-exchange chromatography and DEAE-5PW ion-exchange chromatography. The overall yield of the enzyme was about 50%, representing approximately a 1000-fold purification.
This exoenzyme displayed optimum activity at pH 5.0 and 42°C. A Kₘ value of 1.92 mM was obtained when the enzyme was assayed with p-nitrophenyl phosphate as the substrate. The enzyme was completely inactivated by 2.5 x 10⁻³ mM ammonium molybdate, 0.1 mM sodium fluoride and 2 .0 mM L-tartrate . Kinetic analysis of the inhibition showed that the ammonium molybdate and L-tartrate were competitive inhibitors of the enzyme, exhibiting a K; value of 6.0 nM and 2.55 uM, respectively. In contrast, sodium fluoride inhibited the acid phosphatase in a complex fashion.
The extracellular acid phosphatase appeared to be heterogeneous upon electrophoresis in both nondissociating and dissociating polyacrylamide gels, suggesting a high degree of post-translational modification. The enzyme was subsequently found to be a phosphorylated glycoprotein by metabolic labelling, binding to Con-A and carbohydrate staining of the SDS-PAGE gels. A molecular weight of 145 K for the major component of this exoenzyme was estimated by SDS-P AGE. The amino acid composition was determined. An earlier determined N-terminal sequence was corroborated and two internal peptide sequences were also established. Carbohydrate compositional analysis provided evidence that the extracellular acid phosphatase contained N-linked oligosaccharides as well as O-linked oligosaccharides which were partially characterized by exoglycosidase digestions, HF-dephosphorylation and Bio-Gel P-4 chromatography after release by hydrazinolysis and reduction with NaB³H₄. The major fragment, which accounted for more than 90% of the total radioactive materials, was found to be a phosphorylated galactosyl-β-mannitol disaccharide. In addition, the charged nature of the oligosaccharides was demonstrated to be exclusively due to phosphate groups. These cumulative chemical results are in agreement with earlier immunological data which inferred that the extracellular acid phosphatase contained a lipophosphoglycan-like phosphoglycan structure.
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UN SDG 6: Clean Water and Sanitation