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Partial purification and characterization of a soluble protein phosphatase fromLeishmania donovani promastigotes

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Abstract

A soluble protein phosphatase from the promastigote form of the parasitic protozoanLeishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns. The partially purified enzyme showed a native molecular weight of about 42, 000 in both Sephadex G-100 and sucrose density gradient centrifugation. The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively. The enzyme preferentially utilized phosphohistone as the best exogenous substrate. Mg2+ ions were essential for enzyme activity; among other metal ions Mn2+ can replace Mg2+ to a certain extent whereas Ca2+, Co2+ and Zn2+ could not substitute for Mg2+. The pH optimum of the enzyme was 6.5–7.5 and the temperature optimum 37°C. The apparent Km for phosphohistone was 7.14 μM. ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid. These results suggest thatL. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.

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Abbreviations

PMSF:

phenylmethylsulfonyl fluoride

DTT:

dithiothreitol

TCA:

trichloroacetic acid

BSA:

bovine serum albumin

EDTA:

ethylenediamine tetraacetic acid

ATP:

adenosine triphosphate

ADP:

adenosine diphosphate

AMP:

adenosine monophosphate

EGTA:

Ethyleneglycol-bis-(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid

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  1. Leishmania Group, Department of Cell Biology, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, 700 032, Calcutta, India

    Srabani Nandi & Dwijen Sarkar

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  1. Srabani Nandi
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Nandi, S., Sarkar, D. Partial purification and characterization of a soluble protein phosphatase fromLeishmania donovani promastigotes. Mol Cell Biochem 148, 191–198 (1995). https://doi.org/10.1007/BF00928156

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