Three forms of glutathione transferase (GST) with pI values of 6.0, 6.4 and 7.3 were isolated from Proteus mirabilis AF 2924 by glutathione-affinity chromatography followed by isoelectric focusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide-slab-gel electrophoresis, all forms proved to be composed of two subunits of identical (22,500) Mr. GST-6.0 and GST-6.4 together account for about 95% of the total activity, whereas GST-7.3 is present only in trace amounts. Extensive similarities have been found between GST-6.0 and GST-6.4. These include subunit molecular mass, amino acid composition, substrate specificities and immunological characteristics. GST-7.3 also cross-reacted (non-identity) with antisera raised against bacterial GST-6.0. None of the antisera raised against a number of human, rat and mouse GSTs cross-reacted with the bacterial enzymes, indicating major structural differences between them and the mammalian GSTs. This conclusion is further supported by c.d. spectra.

The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421-425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli sigma 70 consensus promoter and to promoters of P. mirabilis cat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-beta-D-thiogalactopyranoside and growth at 37 degrees C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.

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The dissociation and unfolding of the homodimeric glutathione transferase (GST) Pi from human placenta, using different physicochemical denaturants, have been investigated at equilibrium. The protein transitions were followed by monitoring loss of activity, intrinsic fluorescence, tyrosine exposure, far-u.v. c.d. and gel-filtration retention time of the protein. At low denaturant concentration (less than 1 M for guanidinium chloride and less than 4.5 M for urea), a reversible dissociation step leading to inactivation of the enzyme was observed. At higher denaturant concentrations the monomer unfolds completely. The same unfolding behaviour was also observed with high hydrostatic pressure as denaturant. Our results indicate that the denaturation of GST Pi is a multistep process, i.e. dissociation of the active dimer into structured inactive monomers followed by unfolding.

The fibrillogenic peptide corresponding to the residues 106-126 of the prion protein sequence (PrP 106-126) is largely used to explore the neurotoxic mechanisms underlying the prion disease. However, whether the neuronal toxicity of PrP 106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. The aim of this study was to correlate the structural state of this peptide with its neurotoxicity. Here we show that the two conserved Gly114 and Gly119 residues, in force of their intrinsic flexibility, prevent the peptide assuming a structured conformation, favouring its aggregation in amyloid fibrils. The substitution of both Gly114 and Gly119 with alanine residues (PrP 106-126 AA mutated peptide) reduces the flexibility of this prion fragment and results in a soluble, b-structured peptide. Moreover, PrP 106-126 AA fragment was highly toxic when incubated with neuroblastoma cells, likely behaving as a neurotoxic protofibrillar intermediate of the wild-type PrP 106-126. These data further confirm that the fibrillar aggregation is not necessary for the induction of the toxic effects of PrP 106-126.