PKB inhibition prevents the stimulatory effect of insulin on glucose transport and protein translocation but not the antilipolytic effect in rat adipocytes.

We identified 1-(5 chloronaphthalenesulfonyl)-1H-hexahydro-1, 4-diazepine, also known as ML-9, as a powerful inhibitor of PKB activity in different cells as well as of recombinant PKB. It also inhibits other downstream serine/threonine kinases, such as PKA and p90 S6 kinase, but not upstream tyrosine phosphorylation or PI3-kinase activation in response to insulin. We compared the effects of ML-9 and wortmannin on several insulin-stimulated effects in isolated rat fat cells. Both ML-9 and wortmannin inhibited glucose transport and GLUT4/IGF II receptor translocation to the plasma membrane. In contrast, only wortmannin inhibited the antilipolytic effect and PDE3B activation by insulin. Thus, ML-9 inhibits PKB but not PI3-kinase activation in response to insulin and is useful to differentiate between these effects. Both PI3-kinase and PKB are important for glucose transport and intracellular protein translocation while PKB does not appear to play an important role for the antilipolytic effect or activation of PDE3B in response to insulin.

Regulation of rat glutathione S-transferase A5 by cancer chemopreventive agents: mechanisms of inducible resistance to aflatoxin B1.

The rat can be protected against aflatoxin B1 (AFB1) hepatocarcinogenesis by being fed on a diet containing the synthetic antioxidant ethoxyquin. Evidence suggests that chemoprotection against AFB1 is due to increased detoxification of the mycotoxin by one or more inducible drug-metabolising enzymes. The glutathione S-transferase (GST) isoenzymes in rat liver that contribute to ethoxyquin-induced chemoprotection against AFB1 have been identified by protein purification. This approach resulted in the isolation of several heterodimeric class alpha GST, all of which contained the A5 subunit and possessed at least 50-fold greater activity towards AFB1-8,9-epoxide than previously studied transferases. Molecular cloning and heterologous expression of rat GSTA5-5 has led to the demonstration that it exhibits substantially greater activity for AFB1-8,9-epoxide than other rat transferases. The A5 homodimer can also catalyse the conjugation of glutathione with other epoxides, such as trans-stilbene oxide and 1,2-epoxy-3-(4'-nitrophenoxy)propane, and possesses high catalytic activity for the reactive aldehyde 4-hydroxynonenal. Western blotting has shown that the A5 subunit is not only induced by ethoxyquin but that it is also induced by other cancer chemopreventive agents, such as butylated hydroxyanisole, oltipraz, benzyl isothiocyanate, indole-3-carbinol and coumarin. In addition to GSTA5, we have identified a novel aflatoxin-aldehyde reductase (AFAR) that is similarly induced by ethoxyquin. However, immunoblotting has shown that GSTA5 and AFAR are not always co-ordinately regulated by chemoprotectors. In order to gain a better understanding of the mechanisms responsible for the induction of GSTA5 protein, the GSTA5 gene has been cloned. It was isolated on two overlapping bacteriophage lambda clones and found to be approximately 12 kb in length. The transcriptional start site of GSTA5 has been identified 228 bp upstream from the ATG translational initiation codon. Computer-assisted analysis of the upstream sequence has indicated the presence of a putative antioxidant responsive element (located between -421 and -429 bp) which may be responsible for the induction of GSTA5 by chemopreventive agents.

Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli.

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

Microsomal glutathione transferase: lipid-derived substrates and lipid dependence.

Rat liver microsomal glutathione transferase was found to display glutathione peroxidase activity toward a variety of oxidized lipids. 1-Linoleoyl-2-palmitoyl phosphatidylcholine hydroperoxide, 2-linoleoyl-1-palmitoyl phosphatidylcholine hydroperoxide, 2-linoleoyl-1-palmitoyl phosphatidylethanolamine hydroperoxide, and cholesteryl linoleate hydroperoxide all served as substrates (0.02, 0.04, 0.02, and 0.02 mumol/min mg, respectively). The phospholipid hydroperoxide glutathione peroxidase activity of the enzyme was found not to require detergent and increased when liposomes containing peroxidized phospholipid were fused with liposomes containing microsomal glutathione transferase. Methyl linoleate ozonide serves as a very efficient substrate for the microsomal glutathione transferase. The unactivated and N-ethylmaleimide-activated enzyme displayed specific activities of 0.74 and 5.9 mumol/min mg, respectively. Upon examination of a series of 4-hydroxyalk-2-enals it was found that the catalytic efficiency of the enzyme increases from the 4-hydroxyhept-2-enal up to the 4-hydroxytetradec-2-enal. The specific activities with the various 4-hydroxyalk-2-enals tested varied between 0.28 and 0.95 mumol/min mg. The phospholipid dependence of the microsomal glutathione transferase was examined in proteoliposomes formed by cholate dialysis. Phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, and rat liver microsomal phospholipids could all be used successfully to reconstitute the enzyme. In conclusion, microsomal glutathione transferase can detoxify a number of lipid peroxidation products as well as a fatty acid ozonide. The results imply a protective role for the enzyme under conditions of oxidative stress.

Kinetic studies on rat liver microsomal glutathione transferase: consequences of activation.

Rat liver microsomal glutathione transferase is activated by sulfhydryl reagents and proteolysis. This property varies, however, depending on the combination, concentration and reactivity of the substrates. Thus, a multi-dimensional diagram can be envisioned in which the parameters affecting enzyme activity and activation are visualized. In principle activation could stem from an alteration in enzyme mechanism, transition-state complementarity, product release rate or pH-rate behaviour. These studies appear to rule out these possibilities and an alternate hypothesis is suggested based on the following experiments: (i) alternate substrate diagnosis of the kinetic mechanism of microsomal glutathione transferase indicates a random sequential mechanism. Non-activated and activated enzyme follow the same mechanism by these criteria. (ii) The microsomal glutathione transferase stabilizes a Meisenheimer complex between 1,3,5-trinitrobenzene and glutathione. The formation constants were similar for the unactivated and activated enzyme ((15 +/- 1).10(3) and (14 +/- 1).10(3) M-1, respectively, at pH 8). Inasmuch as the Meisenheimer complex resembles the transition state there is no evidence for an increased stabilization upon activation. (iii) The catalytic rate constant kcat does not vary with the viscosity in the assay medium. Thus, product release is not rate limiting for the unactivated and activated microsomal glutathione transferase (with saturating 1-chloro-2,4-dinitrobenzene and varying GSH). (iv) The pH dependence of the Kf-values for Meisenheimer complex formation exhibited pKa values close to 6 for both the activated and unactivated microsomal glutathione transferase. The pH profile of kcat (with saturating 1-chloro-2,4-dinitrobenzene and variable GSH concentrations) showed apparent pKa values of 5.7 +/- 0.5 and 6.3 +/- 0.4 for the unactivated and activated enzyme, respectively, indicative of a very similar requirement for deprotonation of the enzyme-GSH-1-chloro-2,4-dinitrobenzene complex. (v) Examination of the kinetic parameters (obtained with GSH as the variable substrate against increasingly reactive electrophilic substrates) in Hammett plots shows that the activation mechanism entails a more efficient utilization of GSH. It is suggested that a higher rate of formation of the glutathione thiolate anion occurs in the activated enzyme.

Evidence that rat liver microsomal glutathione transferase is responsible for glutathione-dependent protection against lipid peroxidation.

Evidence that rat liver microsomal glutathione transferase is responsible for the glutathione-dependent inhibition of lipid peroxidation in liver microsomes has been obtained. Activation of the microsomal glutathione transferase in microsomes by cystamine renders this organelle even more resistant to lipid peroxidation in the presence of glutathione compared with untreated microsomes. Upon examining the effect of seven glutathione analogues on lipid peroxidation, it was found that only those that serve as good substrates for the microsomal glutathione transferase (Glutaryl-L-Cys-Gly and alpha-L-Glu-L-Cys-Gly) can inhibit lipid peroxidation. The lack of inhibition by the other five analogues (alpha-D-Glu-L-Cys-Gly, gamma-D-Glu-L-Cys-Gly, beta-L-Asp-L-Cys-Gly, alpha-L-Asp-L-Cys-Gly and alpha-D-Asp-L-Cys-Gly) shows the specificity of the protection and rules out any non-enzymic component. Inhibitors of selenium-dependent glutathione peroxidase (mercaptosuccinate at 50 microM) and phospholipid hydroperoxide glutathione peroxidase (iodoacetate, 1 mM + glutathione, 0.5 mM) do not inhibit the glutathione-dependent protection of rat liver microsomes against lipid peroxidation. Purified microsomal glutathione transferase, NADPH-cytochrome P450 reductase and cytochrome P450 were reconstituted in microsomal phospholipid vesicles by cholate dialysis. The resulting membranes contained functional enzymes and did display enzymic lipid peroxidation induced by 75 microM NADPH and 10 microM Fe-EDTA (2:1). This model system was used to investigate whether microsomal glutathione transferase could inhibit lipid peroxidation in a glutathione-dependent manner. The results show that 5 mM glutathione did inhibit lipid peroxidation when functional microsomal glutathione transferase was included. This was not the case when the enzyme had been pre-inactivated with diethylpyrocarbonate. Furthermore, the protective effect of glutathione could be partly reversed by an inhibitor (100 microM bromosulphophtalein) of the enzyme. Apparently, rat liver microsomal glutathione transferase has the capacity to inhibit lipid peroxidation in a reconstituted system.

Human liver microsomal glutathione transferase. Substrate specificity and important protein sites.

Human liver microsomal glutathione transferase displays the following glutathione peroxidase/transferase activities: dilinoleoylphosphatidylcholine hydroperoxide (0.03 and 0.17 mumol/min.mg, unactivated and N-ethylmaleimide-activated enzyme, respectively), linoleic acid hydroperoxide (0.09 and 0.15 mumol/min.mg), cumene hydroperoxide (0.04 and 3 mumol/min.mg), methyl linoleate ozonide (0.02 and 1.2 mumol/min.mg) and 1-chloro-2,4-dinitrobenzene (1.9 and 24 mumol/min.mg). The activation of glutathione peroxidase activities are much higher than previously observed. The activity towards a phospholipid hydroperoxide is noteworthy since protection against lipid peroxidation has been implied. Methyl linoleate ozonide has not previously been characterised as substrate for any microsomal glutathione transferase. Human liver microsomal glutathione transferase displays an isoelectric point of 9.4 and a structure in agreement with that deduced from the cDNA sequence. Gel electrophoretic analysis shows that proteolytic activation of the human enzyme corresponds to cleavage at Lys-41, thus defining the critical activation site.

Studies on the activity and activation of rat liver microsomal glutathione transferase with a series of glutathione analogues.

The substrate specificity of rat liver microsomal glutathione transferase toward glutathione has been examined in a systematic manner. Out of a glycyl-modified and eight gamma-glutamyl-modified glutathione analogues, it was found that four (glutaryl-L-Cys-Gly, alpha-L-Glu-L-Cys-Gly, alpha-D-Glu-L-Cys-Gly, and gamma-L-Glu-L-Cys-beta-Ala) function as substrates. The kinetic parameters for three of these substrates (the alpha-D-Glu-L-Cys-Gly analogue gave very low activity) were compared with those of GSH with both unactivated and the N-ethylmaleimide-activated microsomal glutathione transferase. The alpha-L-Glu-L-Cys-Gly analogue is similar to GSH in that it has a higher kcat (6.9 versus 0.6 s-1) value with the activated enzyme compared with the unactivated enzyme but displays a high Km (6 versus 11 mM) with both forms. Glutaryl-L-Cys-Gly, in contrast, exhibited a similar kcat (8.9 versus 6.7 s-1) with the N-ethylmaleimide-treated enzyme but retains a higher Km value (50 versus 15 mM). Thus, the alpha-amino group of the glutamyl residue in GSH is important for the activity of the activated microsomal glutathione transferase. These observations were quantitated by analyzing the changes in the Gibbs free energy of binding calculated from the changes in kcat/Km values, comparing the analogues to GSH and each other. It is estimated that the binding energy of the alpha-amino group of the glutamyl residue in GSH contributes 9.7 kJ/mol to catalysis by the activated enzyme, whereas the corresponding value for the unactivated enzyme is 3.2 kJ/mol. The importance of the acidic functions in glutathione is also evident as shown by the lack of activity with 4-aminobutyric acid-L-Cys-Gly and the low kcat/Km values with gamma-L-Glu-L-Cys-beta-Ala (0.03 and 0.01 mM-1s-1 for unactivated and activated enzyme, respectively). Utilization of binding energy from a correctly positioned carboxyl group in the glycine residue (10 and 17 kJ/mol for unactivated and activated enzyme, respectively) therefore also appears to be required for optimal activity and activation. A conformational change in the microsomal glutathione transferase upon treatment with N-ethylmaleimide or trypsin, which allows utilization of binding energy from the alpha-amino group of GSH as well as the glycine carboxyl in catalysis, is suggested to account for at least part of the activation of the enzyme.

Inhibition studies on rat liver microsomal glutathione transferase.

A set of inhibitors for rat liver microsomal glutathione transferase have been characterized. These inhibitors (rose bengal, tributyltin acetate, S-hexylglutathione, indomethacin, cibacron blue and bromosulphophtalein) all have I50 values in the 1-100 microM range. Their effects on the unactivated enzyme were compared to those on the N-ethylmaleimide- and trypsin-activated microsomal glutathione transferase. It was found that the I50 values were decreased upon activation of the enzyme (5-20-fold), except for S-hexylglutathione, where a slight increase was noted. Thus, the activated microsomal glutathione transferase is generally more sensitive to the effect of inhibitors than the unactivated enzyme. It was also noted that inhibitor potency can vary dramatically depending on the substrate used. The I50 values for the N-ethylmaleimide- and trypsin-activated enzyme preparations are altered in a similar fashion compared to the unactivated enzyme. This finding indicates that these two alternative mechanisms of activation induce a similar type of change in the microsomal glutathione transferase.

Activity of rat liver microsomal glutathione transferase toward products of lipid peroxidation and studies of the effect of inhibitors on glutathione-dependent protection against lipid peroxidation.

Rat liver microsomal glutathione transferase displays glutathione peroxidase activity with linoleic acid hydroperoxide, linoleic acid ethyl ester hydroperoxide, and dilinoleoyl phosphatidylcholine hydroperoxide, with rates of 0.2, 0.3, and 0.3 mumol/min/mg, respectively. The activities are increased between three- and fourfold when the enzyme is activated with N-ethylmaleimide. Microsomal glutathione transferase can also conjugate 4-hydroxynon-2-enal with a specific activity of 0.5 mumol/min/mg. These findings show that the enzyme can remove harmful products of lipid peroxidation and thereby possibly protect intracellular membranes against oxidative stress. A set of glutathione transferase inhibitors (rose bengal, tributyltin acetate, S-hexylglutathione, indomethacin, cibacron blue, and bromosulfophtalein) which abolish the glutathione-dependent protection against lipid peroxidation in liver microsomes have been characterized. These inhibitors were found to be effective in the micromolar range and could prove valuable in studying the factor responsible for glutathione-dependent protection against lipid peroxidation.