Monomer−Dimer Equilibrium in Glutathione Transferases: A Critical Re-Examination

Abstract

Glutathione transferases (GSTs) are dimeric enzymes involved in cell detoxification versus many endogenous toxic compounds and xenobiotics. In addition, single monomers of GSTs appear to be involved in particular protein-protein interactions as in the case of the pi class GST that regulates the apoptotic process by means of a GST-c-Jun N-terminal kinase complex. Thus, the dimer-monomer transition of GSTs may have important physiological relevance, but many studies reached contrasting conclusions both about the modality and extension of this event and about the catalytic competence of a single subunit. This paper re-examines the monomer-dimer question in light of novel experiments and old observations. Recent papers claimed the existence of a predominant monomeric and active species among pi, alpha, and mu class GSTs at 20-40 nM dilution levels, reporting dissociation constants (K(d)) for dimeric GST of 5.1, 0.34, and 0.16 microM, respectively. However, we demonstrate here that only traces of monomers could be found at these concentrations since all these enzymes display K(d) values of <<1 nM, values thousands of times lower than those reported previously. Time-resolved and steady-state fluorescence anisotropy experiments, two-photon fluorescence correlation spectroscopy, kinetic studies, and docking simulations have been used to reach such conclusions. Our results also indicate that there is no clear evidence of the existence of a fully active monomer. Conversely, many data strongly support the idea that the monomeric form is scarcely active or fully inactive.

Trim58 Degrades Dynein and Regulates Terminal Erythropoiesis

“…Purified bacterially expressed PS and GST-DIC (1–120) each eluted in gel filtration as a single peak when run individually (Figure 2G). The MALLS data, which provide a reference-free estimate of solution molecular weight, showed that PS eluted as a monomer (observed MW = 25.6 kD; expected mass = 23.3 kD), whereas GST-DIC (1–120) ran as a dimer (observed MW = 91.2 kD; expected mass of dimer = 79.6 kD), reflecting the known dimerization of GST (Fabrini et al, 2009). An equimolar mixture of PS and GST-DIC eluted predominantly as a distinct single peak with a lower retention time, indicating formation of a complex.…”

Glutathione transferases as mediators of signaling pathways involved in cell proliferation and cell death

“…38 This hypothesis would seem to be supported by a recent study showing the dissociation constant of homodimeric GSTP1-1 to be in the subnanomolar range (K d o 1 nM), making it unlikely that the monomeric form would exist in any significant amount at the concentrations commonly used for assay measurement. 39 The interaction between GSTp and JNK has also been established in vivo. Compared with wild-type mice (GSTP1/ P2…”

PDZ affinity chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

“…A potential disadvantage of the PDZ Domain Affinity Chromatography system is that, unlike the His Tag system, it cannot be used under denaturing conditions. When compared to the GST Tag system, the PDZ Domain Affinity System also has advantages: again, the affinity of the tag for the affinity resin is higher (0.1 to 1 μM [13-15] versus 40-170 μM [94-98]); it can be used in the presence of reducing agents; and the affinity tags are monomeric, whereas glutathione is dimeric or multimeric. The PDZ Domain-related Affinity Resins do not show detectable background binding of E. coli host cell proteins [89].…”