setaria cervi: kinetic studies of filarial glutathione synthetase by high performance liquid...
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1. Introduction In most of previous research, emphasis has been giv-
ing the reaction (Beutler and Gelbart, 1986; Seelig andMeister, 1985). These methods can be used on fully/partially puried enzyme, but are unsuitable for crude
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* Corresponding author. Fax: +91 522 2223938/2223405.E-mail address: [email protected] (S. Gupta).
Experimental Parasitology 1110014-4894/$ - see front matter 2005 Elsevier Inc. All rights reservedThe role of GSH as an important antioxidant in lar-ial nematodes is well established (Selkirk et al., 1998). Itprotects lariae from the oxidative stress of ROS pro-duced by the host immune cells, thus aiding their longterm survival in the host body. The ultimate step forGSH biosynthesis is attributed to GS and is well charac-terized in mammals (Gali and Board, 1995; Gali andBoard, 1997; Huang et al., 1995; Luo et al., 2000;Oppenheimer et al., 1979). However, GS has not beencharacterized in nematodes and lariids. The presentstudy is a preliminary step in this direction in supportof future work.
en to studies of larial GCL. This enzyme is well charac-terized in Onchocerca volvulus and Setaria cervi (Luersenet al., 2000; Tiwari et al., 2003). Less emphasis has beenplaced on GS, except for the eect of some syntheticdiglycosylated diaminoalkanes and coumarin deriva-tives in our own studies (Gupta et al., 2004). In the pres-ent work GS has been conrmed as a cytosolic enzymein S. cervi larial worms and its kinetic properties havebeen studied.
Classical methods for GS assay involve quantitativedetermination of inorganic phosphate (Pi) producedduring the reaction or the coupled enzyme procedurewhich allows the formation of ADP to be followed dur-Abstract
The bovine larial worm Setaria cerviwas found to have abundance of glutathione synthetase (GS; EC 6.3.2.3) activity, the enzymebeing involved in catalysing the nal step of glutathione (GSH) biosynthesis. ARP-HPLCmethod involving precolumn derivatizationwith o-phthalaldehyde has been followed for the estimation of GS activity in crude larial preparations. Subcellular fractionation ofthe enzyme was undertaken and it was conrmed to be a soluble protein residing mainly in cytosolic fraction. Attempts to determinethe Km value for L-c-glutamyl-L-cysteine gave a distinctly nonlinear double-reciprocal plot in which data obtained at relatively highdipeptide concentrations (>1 mM) extrapolate to a Km value of about 400 lM whereas data obtained at lower concentrations(