Glutathione-agarose Affinity Chromatography Research Articles

Biochemical properties of cloned glutathione S-transferases from Schistosoma mansoni and Schistosoma japonicum

cDNA clones encoding a 28-kDa subunit glutathione S-transferase (GST) from Schistosoma mansoni (Sm28GST) and a 26-kDa subunit GST from Schistosoma japonicum (Sj26GST) have been expressed in bacterial systems. The recombinant proteins were purified to homogeneity by batch-wash glutathione-agarose affinity chromatography and their biochemical properties investigated. Gel filtration chromatography indicated that both recombinant GSTs are homodimeric proteins. Resolution of Sm28GST and Sj26GST by chromatofocusing in the ranges pH 9–6 and pH 7–4 gave pI estimates of 7.4 and 5.0, respectively. Kinetic analyses suggested that both Sm28GST and Sj26GST operate via a sequential bisubstrate catalytic mechanism. Sm28GST and Sj26GST displayed a mosaic of mammalian Alpha-, Mu- and Pi-type substrate specificities and inhibitor sensitivities. However, multivariate analysis suggests that Sm28GST has an overall catalytic homology with mammalian Mu class GSTs, whilst the enzymatic properties of Sj26GST appear to constitute a hybridisation of Mu and Alpha class features. Both recombinant GSTs interact with a range of hydrophobic ligands including haematin and related compounds, bile acids and several anthelmintics. Sm28GST and Sj26GST possess relatively limited selenium-independent glutathione peroxidase activities, but are able to catalyse the glutathione conjugation of members of the trans,trans-alka-2,4-dienal, trans-alk-2-enal and 4-hydroxyalk-2-enal series of reactive carbonyls (known secondary products of lipid peroxidation).

Fasciola hepatica: Host responders and nonresponders to parasite glutathione S-transferase

Fasciola hepatica glutathione S-transferase (FhGST) was isolated from adult worms by glutathione agarose affinity chromatography. SDS-PAGE shows three proteins of M r ranging from 29-27.8 kDa. Western immunoblot analyses using SDS-PAGE separated adult worm extracts and probed with a rabbit anti-FhGST antiserum reveal two bands in the same M r range. Mice and rabbits immunized with purified FhGST develop copious amounts of anti-FhGST antibodies. Moreover, antisera to F. hepatica adult worms and excretion-secretion products also react with FhGST. Cross-reactivity with schistosomes is evidenced in the reactivity with FhGST of anti- Schistosoma mansoni adult worm antisera and, to a lesser extent, antisera to S. mansoni-soluble egg antigens. The time of appearance of anti-FhGST antibodies in different species of animals infected with F. hepatica was determined. Sheep and a New Zealand white rabbit developed anti-FhGST antibodies detectable by ELISA as early as 2 weeks postexposure with F. hepatica. However, neither mice nor calves infected with F. hepatica developed antibodies to FhGST through the 5–10 weeks of infection tested. But mice infected with S. mansoni developed anti-FhGST cross-reacting antibodies by 6 weeks of infection. Calves immunized with a Fasciola/Schistosoma cross-reactive, cross-protective antigen complex in which a 12,000-kDa protein (Fh12) has been shown to contain immunoprophylactic activity, also developed antibodies to FhGST. Since FhGST is a novel potential vaccine, its protection-inducing capability in a multivalent vaccine combined with Fh12 clearly warrants study. In summary, it appears that hosts with fascioliasis are either responders to FhGST (rabbits, sheep) or nonresponders (mice, cattle), offering interesting models for studying the immune response.

New vectors for high level expression of recombinant proteins in bacteria

A system has been developed for synthesis and rapid purification of recombinant polypeptides expressed in frame with glutathione S-transferase ( D. B. Smith and K. S. Johnson, 1988, Gene 67, 31–40). Expressed fusion proteins are purified from bacterial extracts by glutathione-agarose affinity chromatography. A thrombin protease cleavage site allowed for proteolysis of the fusion protein. We reported the construction of the vector pGEX-KG ( K. Guan and J. E. Dixon, 1991, Anal. Biochem. 192, 262–267) which has a glycine-rich “kinker” immediately after the thrombin cleavage site. This kinker dramatically improved the thrombin cleavage effeiciency of several fusion proteins. One potential drawback of expressing proteins in this vector is that, following proteolytic cleavage, unrelated amino acids from the vector remain at the amino terminus of the released protein. These extensions could affect enzymatic activity or protein structure. We have constructed two new vectors, pGEX-KT and pGEX-KN, which have the glycine kinker placed N-terminal to the thrombin cleavage site in order to minimize the unrelated amino acids associated with the cleaved protein. The change in location of the kinker had no effect on the increased thrombin cleavage efficiency. A strategy combining the kinker in the vector pGEX-KN with polymerase chain reaction has also been developed to express fusion proteins which when cleaved with thrombin released a protein having no amino terminal extensions of any kind.

Leukotriene C4 Synthesis Catalyzed by Dirofilaria Immitis Glutathione S-Transferase

The biologically active sulfidopeptide leukotriene, leukotriene C4, is formed by the enzymatic action of leukotriene C4 synthase, which conjugates glutathione with leukotriene A4. We have found that a filarial glutathione S-transferase can function as a leukotriene C4 synthase. Glutathione S-transferase was purified from the cytosol of adult Dirofilaria immitis by glutathione-agarose affinity chromatography and was reacted with 25 microM leukotriene A4 methyl ester and 10 mM glutathione. The filarial enzyme catalyzed the formation of leukotriene C4 methyl ester, as shown by reverse phase high pressure liquid chromatographic analyses. The finding that filarial glutathione S-transferase can function as leukotriene C4 synthase provides a mechanism whereby filarial parasites could form lipoxygenase pathway derived sulfidopeptide leukotrienes.

Glutathione and glutathione-S-transferase in fungi: Effect of pentachloronitrobenzene and 1-chloro-2,4-dinitrobenzene; Purification and characterization of the transferase from Fusarium

Shortly (3–6 hr) after exposure to either the fungicide pentachloronitrobenzene (PCNB) or 1-chloro-2,4-dinitrobenzene (CDNB) the pool of reduced glutathione (GSH) in Fusarium oxysporum f.sp. melonis and Rhizoctonia solani is drastically depleted, followed by a gradual increase in GSH levels. A similar pattern regarding the effect of the above compounds on glutathione-S-transferase (GST) specific activity was observed in both fungi. There is no significant differences in either GSH levels or GST specific activities in F. oxysporum and R. solani, tolerant and sensitive to PCNB, respectively. The GST was purified from the cytosolic fraction of F. oxysporum using glutathione-agarose affinity chromatography as a major purification step. The enzyme was purified about 753-fold to apparent homogeneity, and its Michaelis-Menten parameters were calculated. Sodium dodecyl sulfate-polyacrylamide electrophoresis revealed one polypeptide of about 25,000 molecular weight.

Isolation and purification of glutathione- S-transferase from the bulb mite Rhizoglyphus robini

Glutathione- S-transferase from the cytosolic fraction of the bulb mite Rhizoglyphus robini has been purified to apparent homogeneity. The purification steps involved DEAE-Sepharose gel chromatography and glutathione-agarose affinity chromatography. The latter resulted in 94-fold purification and in 47% recovery of the enzyme activity present in the cytosol. The K m values for l-chloro-2,4-dinitrobenzene and reduced glutathione were calculated to be 0.13 and 0.26 mM, respectively. Sodium dodecyl sulphate-polyacrylamide electrophoresis revealed one polypeptide band of 25,000 daltons molecular weight.