Partial purification and characterization of a soluble protein phosphatase from Leishmania donovani promastigotes.
A soluble protein phosphatase from the promastigote form of the parasitic protozoan Leishmania 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 degrees C. The apparent Km for phosphohistone was 7.14 microM. 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 that L. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.
Nandi
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Department of Cell Biology, Indian Institute of Chemical Biology, Calcutta, India.
Sarkar
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D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000254:growth & development; D008274:Magnesium / Q000378:metabolism; D008322:Mammals / Q000378:metabolism; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C; D015800:Protozoan Proteins / Q000302:isolation & purification* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D010750:Phosphoproteins; D015800:Protozoan Proteins; D029701:Saccharomyces cerevisiae Proteins; C087358:PTC1 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C; D008274:Magnesium