Purification, characterization, and enzyme kinetics of a glutathione S transferase from larvae of the camel tick Hyalomma dromedarii.

Abstract

Background: Glutathione s-transferases (GSTs) perform an essential role in detoxification of xenobiotics and endogenous compounds via their conjugation to reduce glutathione.

Results: A GST enzyme, designated tick larvae glutathione S transferase (TLGST), was purified from larvae of the camel tick Hyalomma dromedarii via ammonium sulfate precipitation, glutathione-Sepharose affinity column and Sephacryl S-300 chromatography. TLGST-specific activity was found to be 1.56 Umg^-1 which represents 39 folds and 32.2% recovery. The molecular weight of TLGST purified from camel tick larvae was found as 42 kDa by gel filtration. TLGST has a pI value of 6.9 and was found a heterodimeric protein of 28 and 14 kDa subunits as detected on SDS-PAGE. The Lineweaver-Burk plot calculated the km for CDNB to be 0.43 mM with Vmax value of 9.2 Umg^-1. TLGST exhibited its optimal activity at pH 7.9. Co²⁺, Ni²⁺ and Mn²⁺ increased the activity of TLGST while Ca²⁺, Cu²⁺, Fe²⁺ and Zn²⁺ inhibited it. TLGST was inhibited by cumene hydroperoxide, p-hydroxymercuribenzoate, lithocholic acid, hematin, triphenyltin chloride, p-chloromercuribenzoic acid (pCMB), N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), iodoacetamide, EDTA and quercetin. pCMB inhibited TLGST competitively with Ki value of 0.3 mM.

Conclusions: These findings will help to understand the various physiologic conditions of ticks and targeting TLGST could be significant tool for development of prospective vaccines against ticks as a bio-control strategy to overcome the rapid grows in pesticide-resistant tick populations.