Activation of rat liver microsomal glutathione transferase by limited proteolysis.

The activity of rat liver microsomal glutathione transferase is increased by limited tryptic proteolysis; the membrane-bound and purified forms of the enzyme are activated about 5- and 10-fold respectively. The cleavage sites that correlate with this activation were determined by amino acid sequence analysis to be located after Lys-4 and Lys-41. Differences in the relative extent of cleavage at these two sites did not consistently affect the degree of activation. Thus the data support the conclusion that cleavage at either site results in activation. The trypsin-activated enzyme was compared with the form activated with N-ethylmaleimide, which modifies Cys-49. These two differently activated forms were found to have similar kinetic parameters, which differ from those of the unactivated enzyme. The relatedness of the two types of activation is also demonstrated by the observation that microsomal glutathione transferase fully activated by N-ethylmaleimide is virtually resistant to further activation by trypsin. This is the case despite the fact that the N-ethylmaleimide-activated enzyme is much more susceptible to trypsin cleavage at Lys-41 than is the untreated enzyme. The latter observation indicates that activation with N-ethylmaleimide is accompanied by a conformational change involving Lys-41.     

On the binding of bilirubin and its structural analogues to hepatic microsomal bilirubin UDPglucuronyltransferase

Hepatic glucuronidation of the asymmetrical natural bilirubin molecule results in formation of two different positional isomers, bilirubin C-8 monoglucuronide and bilirubin C-12 monoglucuronide. In view of the existence of multiple isoforms of UDPglucuronyltransferase, which is the microsomal enzyme system responsible for bilirubin esterification, we performed kinetic analysis of microsomal glucuronidation of bilirubin and a number of its structural congeners to determine whether synthesis of the two monoglucuronide isomers involved two distinct substrate-binding sites or reflected two different modes of binding to a single catalytic site. Both isomers were found in all tested species (man, rat, guinea pig, sheep), but there were marked species differences in the C-8/C-12 ratio of monoglucuronide found in bile or formed by liver microsomes. Correspondence between in vivo and in vitro results for such regioselectivity of glucuronidation was excellent in each species. On the basis of our results of kinetic analysis of bilirubin esterification at variable pigment substrate concentrations and inhibition studies with alternative substrates, we postulate that both natural monoglucuronide isomers are synthesized at a single binding site. Possible mechanisms responsible for the markedly regioselective esterification of bilirubin by rat and sheep liver were investigated by study of glucuronidation of selected structural analogues of the pigment. Our results do not support explanations of regioselectivity of bilirubin glucuronidation in terms of (i) preferential binding of either the C-8- or C-12-containing dipyrrolic half of the asymmetrical bilirubin molecule or (ii) enantioselective complexation of bilirubin UDPglucuronyltransferase to one of the two chirality enantiomers of intramolecularly hydrogen-bonded bilirubin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation and inhibition of microsomal glutathione transferase from mouse liver.

Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.     

Activation of microsomal glutathione transferase activity by reactive intermediates formed during the metabolism of phenol

The activity of microsomal glutathione transferase was increased 1.7-fold in rat liver microsomes which carried out NADPH dependent metabolism of phenol. Known phenol metabolites were therefore tested for their ability to activate the microsomal glutathione transferase. The phenol metabolites benzoquinone and 1,2,4-benzenetriol both activated the glutathione transferase in microsomes 2-fold independently of added NADPH. However, NADPH was required to activate the enzyme in the presence of hydroquinone. Catechol did not activate the enzyme in microsomes. The purified enzyme was activated 6-fold and 8-fold by 5 mM benzenetriol and benzoquinone respectively. Phenol, catechol or hydroquinone had no effect on the purified enzyme. When microsomal proteins that had metabolized [14C]phenol were examined by SDS polyacrylamide gel electrophoresis and fluorography it was found that metabolites had bound covalently to a protein which comigrated with the microsomal glutathione transferase enzyme. We therefore suggest that reactive metabolites of phenol activate the enzyme by covalent modification. It is discussed whether the binding and activation has general implications in the regulation of microsomal glutathione transferase and, since some reactive metabolites might be substrates for the enzyme, their elimination through conjugation.     

Activation of hepatic microsomal glucuronyltransferase from gunn rats by exposure to light

Exposure of hepatic microsomes from icteric and nonicteric rats to 250 foot candles of blue fluorescent light for 4 hours at 4–6°C significantly increased the activity of UDPglucuronyltransferase activity using p-nitrophenol as a substrate. The light exposure reduced serum bilirubin concentration from icteric rats or bilirubin of fortified human serum albumin by 70 per cent but there was no difference in light activation of hepatic microsomes from icteric or nonicteric rats. Light exposure also decreased the activation of UDPglucuronyltransferase produced by Triton X-100.     

Androgen glucuronyl transferase activity in rat liver, evidence for the importance of hepatic tissue in 5 alpha-reduced androgen metabolism

To elucidate the role of the liver in 5 alpha-reduced androgen metabolism, we used a rat liver glucuronyl transferase assay to determine the conversion of 17 beta-hydroxy-5 alpha-androstane-3-one (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol), and androsterone to their glucuronide metabolites. Serum levels of the two isomers of androstanediol glucuronide (androstanediol 3- and 17-glucuronide) were also measured. Using 5 microM unconjugated steroid as substrate, the production rate (pmol/mg/min) for each product from its respective unconjugated steroid was 6.9 +/- 0.4 for DHT glucuronide, 101 +/- 3.3 for androstanediol 3-glucuronide, 71 +/- 2.0 for androstanediol 17-glucuronide, and 181 +/- 11 for androsterone glucuronide. Production rates for androstanediol glucuronide were 800 times greater for rat liver than for rat prostate, when examined under similar conditions. In the presence of either 0 or 5 microM unlabeled androstanediol, about 60% of the androstanediol glucuronide formed by rat liver was androstanediol 3-glucuronide. In normal male rat serum, 69 +/- 8% (mean +/- SEM) of total androstanediol glucuronide was androstanediol 3-glucuronide. We have previously shown that rat prostate forms androstanediol 17-glucuronide, but not androstanediol 3-glucuronide. The results from the present study indicate that rat liver forms both androstanediol glucuronide isomers, and does so in about the same ratio as is found in rat serum. The rate of glucuronidation is also much greater in rat liver than in rat prostate. While other sites of glucuronidation are possible, these results are consistent with the hypothesis that DHT and other unconjugated androgens formed in rat prostate are conjugated to glucuronic acid mainly in the liver.     

Comparison of hepatic, renal and intestinal bilirubin UDP-glucuronyl transferase activities in rat microsomes

1. Bilirubin UDP-glucuronyltransferase activity and its dependence on substrate concentrations in rat liver, renal cortex and intestinal mucosa microsomes were studied. 2. Bilirubin monoglucuronide synthesis from unconjugated bilirubin was a higher capacity, lower affinity step in comparison with bilirubin diglucuronide formation in the three tissues tested. 3. Bilirubin glucuronide formation in liver microsomes showed a higher capacity but a lower affinity than extrahepatic ones. Renal cortex and intestinal mucosa exhibited similar kinetics parameters. 4. In vitro bilirubin glucuronidation in renal cortex and intestinal mucosa was quantitatively important as compared with the hepatic one.     

Activation of HMG-CoA reductase by microsomal phosphatase

HMG-CoA reductase activity can be modulated by a reversible phosphorylation-dephosphorylation with the phosphorylated form of the enzyme being inactive and the dephosphorylated form, active. Phosphatases from diverse sources, including cytosol, have been shown to dephosphorylate and activate HMG-CoA reductase. The present study demonstrates phosphatase activity capable of activating HMG-CoA reductase that is associated with purified microsomes. The incubation of microsomes at 37 degrees C for 40 min results in a twofold stimulation of HMG-CoA reductase activity, and this stimulation is blocked by sodium fluoride or phosphate. The ability of microsomes to increase HMG-CoA reductase activity occurs regardless of whether microsomes are prepared by ultracentrifugation or calcium precipitation. Additionally, phosphatases capable of activating HMG-CoA reductase are present in both the smooth and rough endoplasmic reticulum. Freeze-thawing does not prevent microsomes from activating HMG-CoA reductase but preincubation results in a significant decrease in the ability of microsomes to increase HMG-CoA reductase activity. Thus, the present study demonstrates that purified liver microsomes contain phosphatase activity capable of activating HMG-CoA reductase.     

Protection of cells from oxidative stress by microsomal glutathione transferase 1

Rat liver microsomal glutathione transferase 1 (MGST1) is a membrane-bound enzyme that displays both glutathione transferase and glutathione peroxidase activities. We hypothesized that physiologically relevant levels of MGST1 is able to protect cells from oxidative damage by lowering intracellular hydroperoxide levels. Such a role of MGST1 was studied in human MCF7 cell line transfected with rat liver mgst1 (sense cell) and with antisense mgst1 (antisense cell). Cytotoxicities of two hydroperoxides (cumene hydroperoxide (CuOOH) and hydrogen peroxide) were determined in both cell types using short-term and long-term cytotoxicity assays. MGST1 significantly protected against CuOOH and against hydrogen peroxide (although less pronounced and only in short-term tests). These results demonstrate that MGST1 can protect cells from both lipophilic and hydrophilic hydroperoxides, of which only the former is a substrate. After CuOOH exposure MGST1 significantly lowered intracellular ROS as determined by FACS analysis.     

Induction of hepatic microsomal calcium uptake by glucocorticoids

Hepatic microsomes take up calcium in the presence of ATP and oxalate. In either fed or fasted adrenalectomized rats injections of dexamethasone 18 hours and then again 1 hour prior to sacrifice increased uptake of calcium by microsomes. Injections of estradiol had no similar effect indicating that the stimulation might be specific to glucocorticoids. Injection of Actinomycin D, an inhibitor of protein synthesis, 1 hour prior to dexamethasone administration resulted in a complete block of the stimulation. It is therefore likely that the increased calcium uptake is due to the induction of the microsomal calcium activated ATP-ase. The onset of this effect occurred later than the induction of tyrosine-amino transferase (TAT). The present data, in conjunction with the previous demonstration of glucagon stimulation and insulin inhibition of this system, indicates that microsomes might serve as a modifier of intracellular calcium distribution.     

NADPH dependent activation of microsomal glutathione transferase 1

Microsomal glutathione transferase 1 (MGST1) can become activated up to 30-fold by several mechanisms in vitro (e.g. covalent modification by reactive electrophiles such as N-ethylmaleimide (NEM)). Activation has also been observed in vivo during oxidative stress. It has been noted that an NADPH generating system (g.s.) can activate MGST1 (up to 2-fold) in microsomal incubations, but the mechanism was unclear. We show here that NADPH g.s treatment impaired N-ethylmaleimide activation, indicating a shared target (identified as cysteine-49 in the latter case). Furthermore, NADPH activation was prevented by sulfhydryl compounds (glutathione and dithiothreitol). A well established candidate for activation would be oxidative stress, however we could exclude that oxidation mediated by cytochrome P450 2E1 (or flavine monooxygenase) was responsible for activation under a defined set of experimental conditions since superoxide or hydrogen peroxide alone did not activate the enzyme (in microsomes prepared by our routine procedure). Actually, the ability of MGST1 to become activated by hydrogen peroxide is critically dependent on the microsome preparation method (which influences hydrogen peroxide decomposition rate as shown here), explaining variable results in the literature. NADPH g.s. dependent activation of MGST1 could instead be explained, at least partly, by a direct effect observed also with purified enzyme (up to 1.4-fold activation). This activation was inhibited by sulfhydryl compounds and thus displays the same characteristics as that of the microsomal system. Whereas NADPH, and also ATP, activated purified MGST1, several nucleotide analogues did not, demonstrating specificity. It is thus an intriguing possibility that MGST1 function could be modulated by ligands (as well as reactive oxygen species) during oxidative stress when sulfhydryls are depleted.     

The projection structure of microsomal glutathione transferase.

Through the use of electron crystallography, it has been possible to obtain high resolution structural information regarding a mammalian protein that spans the lipid bilayer. Two-dimensional crystals of the detoxification enzyme microsomal glutathione transferase were induced by slow detergent removal from a mixture containing low amounts of phospholipid. Images of specimens stabilized in tannin were collected using electron cryomicroscopy. The projection structure at 4 A shows tightly packed trimers of the protein. Each of them contains an inner core of six parallel alpha-helices delineating a central low density region. The helical bundle is partly surrounded by elongated domains.     

Hepatic microsomal bilirubin UDP-glucuronosyltransferase. The kinetics of bilirubin mono- and diglucuronide synthesis.

Hepatic biotransformation of bilirubin to the hydrophilic species bilirubin mono- (BMG) and diglucuronide (BDG) by microsomal bilirubin UDP-glucuronosyl-transferase (GT) is a prerequisite for its physiologic excretion into bile. The reaction mechanism of bilirubin-GT and the access of bilirubin and BMG (the intermediate substrate) to the active site of bilirubin-GT are undefined. Highly purified [14C]bilirubin and [3H] BMG were coincubated with rat liver microsomes, and the initial rates of radiolabeled bilirubin glucuronide synthesis were measured. Although these substrates differ markedly in their hydrophilicity, no significant differences were observed in [14C]- and [3H]BDG rates of formation from equimolar [14C]bilirubin and [3H] BMG, in the absence or presence of soluble binding proteins (albumin and hepatic cytosol). In further kinetic studies, [14C]bilirubin and [3H]BMG exhibited mutually competitive inhibition of [3H]- and [14C]BDG synthesis, respectively, and [3H]BMG also inhibited [14C]BMG formation. Finally, unlabeled BMG and BDG inhibited the glucuronidation of [14C]bilirubin, with all three pigments yielding virtual Michaelis-Menten dissociation constants in the 10-20 microM range. These findings indicate that: 1) bilirubin-GT follows Michaelis-Menten kinetics for both bilirubin and BMG glucuronidation over the range of substrate concentrations employed; 2) the findings are consistent with a single active site for the enzymatic synthesis of both BMG and BDG; 3) bilirubin, BMG, and BDG bind competitively to this active site with comparable affinities; and 4) access of both bilirubin and BMG substrates to the enzymatic active site is reduced by soluble binding proteins.