Released August 2007 - The Indian Society for Parasitology

34Sharma and Banyalcarried out recombinant expression and min; 10,000 g, 25 min; 24,000 g, 35 min and 1,05,000characterization of the protein. P. berghei, a rodent g, 60 min. The pellets were suspended, separately, inmalaria parasite, maintains sustainable GSH homogenization buffer, homogenized if required andconcentration and survives in host erythrocytes. The assayed along with cytosolic fraction.parasite possesses significant GS activity mainlyconfined to its cytosolic part, which in the presentPurification of GS: P. berghei GS was purifiedstudy, has been purified through columnaccording to a slightly modified method of Meisterchromatography and characterized.(1985). Homogenate of cell-free P. berghei parasiteswas centrifuged at 450 g for 10 min, and theMATERIALS AND METHODSsupernatant first subjected to ammonium sulphateprecipitation at 0-35%, 35-70% and 70-100%Cell-free parasites: Plasmodium berghei (NK-65)saturation with solid ammonium sulphate. Thewas maintained in albino Swiss mice (Banyal et al.,fractions were suspended in buffer A and anlaysed for1991). Blood, collected from normal or infected mice,GS activity.was passed through CF-11 cellulose column and lysedwith 0.2% (w/v) saponin in 0.01M PBS (pH7.2). Total Gel filteration chromatography on Sephadex G-erythrocytes and their fractions haemolysate, 200: Dried Sephadex G-200 (2 g; Sigma) was used forerythrocyte membranes and cell-free parasites were a 50 ml bed volume. The gel was swollen in distilledcollected as previously described (Kumar and Banyal, water at room temperature overnight. After swelling, it1997). was loaded onto a glass column (2 cm x 16 cm). Flowrate was adjusted to 15 ml/h. The column equilibratedGS (γ-L-glutamate-L-cysteine: glycine ligase EC with 50 mM imidazole-HCl buffer (pH 7.5) until the6.3.2.3) assay: Normal and P. berghei-infected final absorbance difference became zero at 280 nmerythrocytes, their fractions and cell-free P. berghei and pH value similar to that of equilibration buffer.were suspended in buffer A containing 50 mM Ammonium sulphate precipitated fraction containingimidazole HCl (pH 7.5), 10 mM magnesium chloride, the enzyme activity was loaded onto the column,0.1 mM phenylmethylsulphonylfluoride, eluted with imidazole-HCl and 45 fractions of 1 ml°homogenized, centrifuged (450 g, 10 min, 4 C) and the each were collected.supernatants used as enzyme extracts. GS was assayedaccording to Meister (1985). The standard 1 ml assay Sodium dodecyl sulphate-polyacrylamide gelmixture contained 100 mM tris HCl (pH 8.2), 1 mM γ- electrophoresis (SDS-PAGE): Purified fractionsL-glutamyl-L-cysteine, 15 mM glycine, 10 mM were characterized by SDS-PAGE according to theadenosine triphosphate (ATP), 10 mM magnesium method of Laemmli (1970) as given by Banyal andchloride, 1 mM phosphoenolpyruvate, 0.24 mM Inselberg (1985) by using 3% stacking and 10%nicotinamide adeninedinucleotide (NADH), 1 unit separating gels. Gels were stained overnight in 0.2%lactate dehydrogenase, 5 units pyruvate kinase, 100 Coomassie brilliant blue R-250.mM potassium chloride and appropriate volume ofenzyme extract. Reaction was initiated by the additionRESULTSof enzyme extract, and decrease in absorbance at 340 P. berghei contained significant amount of GS activitynm was monitored spectrophotometrically (Perkin (Table I). Parasitized host erythrocytes possessedElmer UV/VIS spectrometer Lambda 12). Protein was about three-times higher enzyme activity thandetermined using folin phenol reagent (Lowry et al., erythrocytes from normal mice. Lysis of erythrocytes1951) and bovine serum albumin was used as a revealed that the enzyme was mainly confined to thestandard. One unit of GS activity is the amount of haemolysate fraction compared to erythrocyteenzyme that catalyzes the formation of one µ mol of membranes. However, the values of GS activity were-1glutathione/hour (ε=6.22 mM ). Specific activity was higher in fractions of parasitized red cells than theexpressed as enzyme units/mg of protein.fractions of normal red cells. Cell-free parasitesdemonstrated approximately 0.443 U/mg of GSSubcellular fractionation: Freshly isolated cell-free activity, which was about 5.6-fold higher than theP. berghei parasites were suspended in 0.25 M sucrose normal host erythrocytes.in 0.01 M PBS and fractionated according to themethod of Banyal et al. (1979). Differential Differential centrifugation of P. berghei resulted incentrifugation of homogenate was carried at 600 g, 15 various sediments and cytosolic (supernatant of g