Biochemical characterization and peptide mass fingerprinting of two glutathione transferases from Biomphalaria alexandrina snails (Gastropoda: Planorbidae).

BACKGROUND: The freshwater snails Biomphalaria alexandrina (Gastropoda: Planorbidae) has public health importance of being an intermediate host of Schistosoma mansoni, the parasite species that causes intestinal schistosomiasis in humans. Glutathione transferases (GSTs) play an important role in detoxification of a broad range of compounds including secondary metabolites and exogenous compounds. Studying GSTs in snails may clarify their role in detoxification of molluscicides. RESULTS: Two glutathione transferases (BaGST2 and BaGST3) were purified and characterized from B. alexandrina snails. BaGST2 and BaGST3 were electrophoretically homogeneous preparations with subunit molecular weight of 23.6 kDa and molecular weight of 45 kDa. Isoelectric focusing of BaGST2 revealed the presence of two components at pI 4.47 and 4.67, while BaGST3 showed one band at pI 4.17. The specific activity of BaGST2 and BaGST3 toward 1-chloro-2,4-dinitrobenzene (CDNB) was 19.0 and 45.2 µmol/min/mg protein following 146- and 346-fold purification, respectively. The catalytic pH optima, km values, and the activation energies for BaGST2 and BaGST3 were determined. BaGST2 and BaGST3 were significantly inhibited by hematin and Cibacron Blue and to a less extent by bromosulfophthalein, S-butyl-GSH, S-hexyl-GSH, and S-P-bromobenzyl-GSH. BaGST2 and BaGST3 showed high activity against ethacrynic acid as substrate, and they also exhibited peroxidase activity on cumene hydroperoxide. The two enzymes showed identical patterns of lysine-C digestion after high-performance liquid chromatography. The amino acid sequences of three peptide fragments and peptide mass fingerprinting of fourteen peptides were used to predict the primary structure of BaGST2. A polypeptide of 206 amino acids (with 7 gaps, 3 of which could not identified) was predicted for BaGST2. The theoretical subunit molecular weight of BaGST2 is 22.6 kDa, with pI of 8.58. BaGST2 has 65% sequence identity and 78% positive with Biomphalaria glabrata GST7. The overall structure of BaGST2 at the N-terminal domain is identical to the canonical GST N-terminal domain, having the typical thioredoxin-like fold with a ßαß-α-ßßα motif, whereas the C-terminal domain is made from 6 α-helices. A conservative GST-N-domain includes glutathione binding sites Y11, L17, Q53, M54, Q65, and S66, while a variable GST-C domain contains electrophilic substrate binding site H99, R102, A103, F106, K107, L161, and Y167. Phylogenetic tree showed that BaGST2 was clustered in the sigma group with GSTs sigma class from invertebrates and vertebrates. CONCLUSIONS: We have purified and characterized two GSTs from B. alexandrina snails. Our study broadens the biochemical information on freshwater snail GSTs by demonstrating the role of BaGSTs in defense mechanisms against structurally different electrophilic compounds. BaGST2 and BaGST3 have Se-independent peroxidase activity, which indicates their role in cellular antioxidant defense by reducing organic hydroperoxides in B. alexandrina. A polypeptide chain of 206 amino acids was predicted. The primary structure of BaGST2 showed 65% sequence identity with Biomphalaria glabrata GST7. Sequence analysis indicates that BaGST2 is a GST-N-sigma-like with a thioredoxin-like superfamily. Phylogenetic tree confirms that BaGST2 belongs to the sigma class of GSTs superfamily.

Purification and biochemical characterization of glutathione S-transferase from Down syndrome and normal children erythrocytes: a comparative study.

Down syndrome (DS) is the phenotypic manifestation of trisomy 21. Our study was concerned with the characterization and purification of glutathione S-transferase enzyme (GST) from normal and Down syndrome (DS) erythrocytes to illustrate the difference in the role of this enzyme in the cell. Glutathione S-transferase and glutathione (GSH) was determined in ten DS and ten healthy children matched for age (3-10 years). DS group exhibited significantly lower GST value (2.7 units/gHb) as compared to controls (6.6 units/gHb) (40.9%). GST activity was significantly decreased to 40.9% in the DS group as compared to controls. Also GSH concentration was significantly decreased to 60.6% in the DS group compared to the controls. Glutathione transferase was purified from erythrocytes of normal and DS pooled blood samples by affinity chromatography with specific activity of 23.7% and 7.9%, respectively. The effect of freezing and thawing, storage time of freezing and GSH concentration on the stability of the enzyme were examined. Normal GST exhibited a pH optimum at pH 7 followed by sharp decrease, however DS GST exhibited pH optimum between pH 7.5 and 8. The Km values for 1-chloro-2,4-dinitrobenzene (CDNB) and GSH were 0.205 mM and 0.786 mM, respectively, for normal GST, and 0.318 mM and 1.307 mM, respectively for DS GST. The activation energy (Ea) was calculated to be 2.25 and 4.25 cal/mol for normal GST and 3.8 cal/mol for DS GST. Normal and DS GST were inhibited by the same inhibitors (hematin, bromosulfophthalein and cibacron blue), but with different degree. On kinetic basis, the individuals with lower overall GST activity and slight differences in some kinetic characters are at greater risk from xenobiotic contamination as compared to those with higher overall GST activity observed in normal individuals.

Multiple unfolding states of glutathione transferase from Physa acuta (Gastropoda [correction of Gastropada]: Physidae).

The equilibrium unfolding of the major Physa acuta glutathione transferase isoenzyme (P. acuta GST(3)) has been performed using guanidinium chloride (GdmCl), urea, and acid denaturation to investigate the unfolding intermediates. Protein transitions were monitored by intrinsic fluorescence. The results indicate that unfolding of P. acuta GST(3) using GdmCl (0-3.0M) is a multistep process, i.e., three intermediates coexist in equilibrium. The first intermediate, a partially dissociated dimer, exists at low GdmCl concentration (approximately at 0.7M). At 1.2M GdmCl, a dimeric intermediate with a compact structure was observed. This intermediate undergoes dissociation into structural monomers at 1.75M of GdmCl. The monomeric intermediate started to be completely unfolding at higher GdmCl concentrations (>1.8M). Unfolding using urea (0-7.0M) and acid-induced structures as well as the fluorescence of 8-anilino-1-naphthalenesulfonate in the presence of different GdmCl concentrations confirmed that the unfolding is a multistep process. At concentrations of GdmCl or urea less than the midpoints or at the midpoint pH (pH 4.2-4.6), the unfolding transition is protein concentration independent and involved a change in the subunit tertiary structure yielding a partially active dimeric intermediate. The binding of glutathione to the enzyme active site stabilizes the native dimeric state.

Two glutathione S-transferase isoenzymes purified from Bulinus truncatus (Gastropoda: Planorbidae).

We purified and characterized two major glutathione S-transferase isoenzymes (GST2 and GST3) from snail Bulinus truncatus (Mollusca, Gastropoda, Planorbidae) tissue. The Km with respect to 1-chloro-2, 4-dinitrobenzene (CDNB) for both isoenzymes was increased as the pH decreased. Km of both isoenzymes with respect to glutathione (GSH) doubled when the pH was increased from 6.0 to 6.5. Acid inactivated GST2 and GST3 and the two enzymes were almost inactive at pH 3.5. However, they retain the full activity for at least 20 h when incubated at pH between 6.0 and 9.0. The optimum temperature was 45 degrees C for GST2 and 50 degrees C for GST3. The half lifetime at 50 degrees C was 70 min and 45 min for GST2 and GST3 isoenzymes, respectively. Addition of 5 mM GSH to the incubation buffer increased the half life of both isoenzymes more than fourfold. The activation energy for catalyzing the conjugation of CDNB was 1.826 and 3.435 kcal/mol for GST2 and GST3, respectively. I50 values for Cibacron blue, bromosulphophthalein, indocyanine green, hematin and ethacrynic acid were 0.76 microM, 47.9 microM, 7.59 microM, 0.03 microM and 0.79 microM for GST2, and 0.479 microM, 79.4 microM, 89.1 microM, 32.4 microM and 1.15 microM for GST3, respectively. Cibacron blue and indocyanine green were non-competitive inhibitors, while hematin was a mixed inhibitor. Bromosulphophthalein was found to be a competitive inhibitor for GST2 and a mixed inhibitor for GST3.

Purification and characterization of a glutathione S-transferase from Mucor mucedo.

An intracellular glutathione transferase was purified to homogenity from the fungus, Mucor mucedo, using DEAE-cellulose ion-exchange and glutathione affinity chromatography. Gel filtration chromatography and SDS-PAGE revealed that the purified GST is a homodimer with approximate native and subunit molecular mass of 53 kDa and 23.4 kDa, respectively. The enzyme has a pI value of 4.8, a pH optimum at pH 8.0 and apparent activation energy (Ea) of 1.42 kcal mol(-1). The purified GST acts readily on CDNB with almost negligible peroxidase activity and the activity was inhibited by Cibacron Blue (IC50 0.252 microM) and hematin (IC50 3.55 microM). M. mucedo GST displayed a non-Michaelian behavior. At low (0.1-0.3 mM) and high (0.3-2 mM) substrate concentration, Km (GSH) was calculated to be 0.179 and 0.65 mM, whereas Km(CDNB) was 0.531 and 11 mM and k(cat) was 39.8 and 552 s(-1), respectively. The enzyme showed apparent pKa values of 6-6.5 and 8.0.

Design of a monomeric human glutathione transferase GSTP1, a structurally stable but catalytically inactive protein.

By the introduction of 10 site-specific mutations in the dimer interface of human glutathione transferase P1-1 (GSTP1-1), a stable monomeric protein variant, GSTP1, was obtained. The monomer had lost the catalytic activity but retained the affinity for a number of electrophilic compounds normally serving as substrates for GSTP1-1. Fluorescence and circular dichroism spectra of the monomer and wild-type proteins were similar, indicating that there are no large structural differences between the subunits of the respective proteins. The GSTs have potential as targets for in vitro evolution and redesign with the aim of developing proteins with novel properties. To this end, a monomeric GST variant may have distinct advantages.