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Biomedical subjects

Ralf Morgenstern

Publications and source records attributed to Ralf Morgenstern.

At least 19 recordsLinked to original sources
PubMed2006

Microsomal glutathione transferase 1 in anticancer drug resistance.

Glutathione transferases (GSTs) are often upregulated in tumors and have been suggested to play an important role in multiple drug resistance in cancer chemotherapy. As a consequence GST-dependent pro-drugs and inhibitors are being developed. Little is known, however, on the potential role of membrane-bound GSTs in drug resistance despite the fact that detoxication of cytostatic drugs and upregulation in tumors has been demonstrated. Therefore, we have studied the involvement of membrane-bound microsomal GST1 (MGST1) in cellular resistance to anticancer drugs. As a tool we have developed a cell system utilizing MCF7 cells stably overexpressing MGST1. Here, we show for the first time that MGST1 can protect cells from several cytostatic drugs, chlorambucil, melphalan and cisplatin in an acute toxicity test (MTT assay) as well as a long-term colony forming efficiency cytotoxicity test. It is of note that these cells do not overexpress multidrug transporters, a prerequisite for protection with certain other GSTs investigated in this system. The cytostatic drugs used comprise both those that are known/predicted to be substrates as well as non-substrates. Thus, the mechanism most probably entails both direct detoxication and downstream protection of the cells from oxidative stress.

Antineoplastic Agents↗
PubMed2006

Structural basis for detoxification and oxidative stress protection in membranes.

Synthesis of mediators of fever, pain and inflammation as well as protection against reactive molecules and oxidative stress is a hallmark of the MAPEG superfamily (membrane associated proteins in eicosanoid and glutathione metabolism). The structure of a MAPEG member, rat microsomal glutathione transferase 1, at 3.2 A resolution, solved here in complex with glutathione by electron crystallography, defines the active site location and a cytosolic domain involved in enzyme activation. The glutathione binding site is found to be different from that of the canonical soluble glutathione transferases. The architecture of the homotrimer supports a catalytic mechanism involving subunit interactions and reveals both cytosolic and membraneous substrate entry sites, providing a rationale for the membrane location of the enzyme.

Amino Acid Sequence↗
PubMed2005

Microsomal glutathione transferase 1.

Microsomal glutathione transferase 1 (MGST1) is an abundant membrane-bound glutathione transferase and peroxidase constituting 3% of the endoplasmic reticulum protein in rat liver (and 5% of the outer mitochondrial membrane). The enzyme is most well studied in mammals and belongs to a large and widely distributed superfamily. Cellular and organelle protection versus oxidative stress has been demonstrated. The enzyme displays activity to a multitude of reactive substrates ranging from products of lipid peroxidation to cytostatic drugs. The methods developed for the study of MGST1 by necessity differs from that of cytosolic glutathione transferases, because detergents or lipids are included. Here, purification, assay, and preparation procedures that maintain the enzyme in its native functional state during isolation and characterization are described. Microsomal glutathione transferase 1 is activated by sulfhydryl reagents (and proteolysis), and procedures for activation and study of the activated enzyme are described. In new developments, the enzyme is studied by pre-steady state methods, as well as mass spectrometry involving direct observation of the native enzyme.

Animals↗
PubMed2005

Bioinformatic and enzymatic characterization of the MAPEG superfamily.

The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belonging to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calculations of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal glutathione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4), 5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2/FLAP/LTC4 synthase families. The insect members are most similar to MGST1/prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, showing an early origin for MAPEG family differentiation. Thus, the evolutionary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the subfamilies. We have further investigated and functionally characterized representative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity analysis demonstrated that all proteins catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 micromol.min(-1).mg(-1) in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N-terminal amino acid sequence that was at least 50% pure, with a specific activity towards 1-chloro-2,4-dinitrobenzene of 11 micromol.min(-1).mg(-1). Yeast microsomes expressing the Arabidopsis enzyme showed an activity of 0.02 micromol.min(-1).mg(-1), whereas the Drosophila enzyme expressed in E. coli was highly active at 3.6 micromol.min(-1).mg(-1). The purified pike enzyme is the most active MGST described so far with a specific activity of 285 micromol.min(-1).mg(-1). Drosophila and pike enzymes also displayed glutathione peroxidase activity towards cumene hydroperoxide (0.4 and 2.2 micromol.min(-1).mg(-1), respectively). Glutathione transferase activity can thus be regarded as a common denominator for a majority of MAPEG members throughout the kingdoms of life whereas glutathione peroxidase activity occurs in representatives from the MGST1, 2 and 3 and PGES subfamilies.

Animals↗
PubMed2004

Stress sensor triggers conformational response of the integral membrane protein microsomal glutathione transferase 1.

Microsomal glutathione (GSH) transferase 1 (MGST1) is a trimeric, integral membrane protein involved in cellular response to chemical or oxidative stress. The cytosolic domain of MGST1 harbors the GSH binding site and a cysteine residue (C49) that acts as a sensor of oxidative and chemical stress. Spatially resolved changes in the kinetics of backbone amide H/D exchange reveal that the binding of a single molecule of GSH/trimer induces a cooperative conformational transition involving movements of the transmembrane helices and a reordering of the cytosolic domain. Alkylation of the stress sensor preorganizes the helices and facilitates the cooperative transition resulting in catalytic activation.

Amino Acid Sequence↗
PubMed2004

Kinetic characterization of thiolate anion formation and chemical catalysis of activated microsomal glutathione transferase 1.

Microsomal glutathione transferase 1 (MGST1) displays the unique ability to be activated, up to 30-fold, by the reaction with sulfhydryl reagents, e.g., N-ethylmaleimide. Analysis of glutathione (GSH) thiolate formation, which occurs upon mixing activated MGST1 with GSH, reveals biphasic kinetics, where the rapid phase dominated at higher GSH concentrations. The kinetic behavior suggests a two-step mechanism consisting of a rapid GSH-binding step (K(D)(GSH) approximately 10 mM), followed by slower formation of thiolate (k(2) approximately 10 s(-1)). The release rate (or protonation of the enzyme GSH thiolate complex) of GS(-) was slow (k(-2) = 0.016 s(-1)), consistent with overall tight binding of GSH. Electrophilic second substrates react rapidly with the E*GS(-) complex, and again, a two-step mechanism is suggested. In comparison to the unactivated enzyme [Morgenstern et al. (2001) Biochemistry 40, 3378-3384], the mechanisms of GSH thiolate formation and electrophile interaction are similar; however, thiolate anion formation is enhanced 30-fold in the activated enzyme, contributing to an increased k(cat) (3.6 s(-1)). Interestingly, in the activated enzyme, thiolate formation and proton release from the enzyme are not strictly coupled, because proton release (as well as k(cat)) was found to be approximately 4 times slower than GSH thiolate formation in an unbuffered system. Solvent kinetic isotope effect measurements demonstrated a 2-fold decrease in the rate constant (k(2)) for thiolate formation and k(cat) (in the reaction with 1-chloro-2,4-dinitrobenzene) for both unactivated and activated MGST1. This indicates that thiolate formation contributes to k(cat) for the activated enzyme, as suggested previously for unactivated MGST1. The stoichiometry of thiolate formation, proton release, and burst kinetics suggested utilization of one GSH molecule per enzyme trimer.

Animals↗
PubMed2004

Structural organization of the murine microsomal glutathione S-transferase gene (MGST1) from the 129/SvJ strain: identification of the promoter region and a comprehensive examination of tissue expression.

The structure and regulation of the murine microsomal glutathione transferase gene (MGST1) from the 129/SvJ strain is described and demonstrates considerable difference in nucleotide sequence and consequently in restriction enzyme sites as compared to other mouse strains. A comparison of the amino acid sequence for MGST1 revealed one difference in exon 2 between the 129/SvJ strain (arginine at position 5) and the sequence previously reported for the Balb/c strain (lysine). The promoter region immediately upstream of the dominant first exon is functional, transcriptionally responds to oxidative stress, and is highly homologous to the human region. Oxidative stress also induced the production of endogenous MGST1 mRNA. The tissue-specific expression of MGST1 mRNA was studied, and as anticipated, was indeed highest in liver. There was, however, marked mRNA expression in several tissues not previously studied including smooth muscle, epidymus, ovaries, and endocrine glands in which the expression of various peroxidases is also very high (salivary and thyroid). Overall, there was a good agreement between the mRNA content detected and previous reports of MGST1 activity with the exception of brain tissue.

Animals↗
PubMed2004

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.

Animals↗
PubMed2004

Amino acid residue 247 in canine sulphotransferase SULT1D1: a new determinant of substrate selectivity.

The SULT (sulphotransferase) family plays a critical role in the detoxification and activation of endogenous and exogenous compounds as well as in the regulation of steroid hormone actions and neurotransmitter functions. The structure-activity relationships of the human SULTs have been investigated with focus on the amino acid 146 in hSULT1A3 and its impact on dopamine/PNP (p-nitrophenol) specificity. In the present study, we have generated canine SULT1D1 (cSULT1D1) variants with mutations at amino acid residues in the substrate-binding pocket [A146E (Ala-146-->Glu), A146D, A146Q, I86D or D247L]. These mutation sites were chosen with regard to their possible contribution to the marked dopamine/PNP preference of cSULT1D1. After characterization, we found that the overall sulphation efficiencies for the cSULT1D1 A146 and the I86 mutants were strongly decreased for both substrates compared with wild-type cSULT1D1 but the substrate preference was unchanged. In contrast, the D247L mutant was found to be more than 21-fold better at sulphating PNP (120-fold decrease in K(m) value) but 54-fold less efficient in sulphating dopamine (8-fold increase in K(m) value) and the preference was switched from dopamine to PNP, indicating the importance of this amino acid in the dopamine/PNP preference in cSULT1D1. Our results show that Asp-247 has a pronounced effect on the substrate specificity of cSULT1D1 and thus we have identified a previously unrecognized contributor to active-site selectivity.

Amino Acids↗
PubMed2004

Observation of an intact noncovalent homotrimer of detergent-solubilized rat microsomal glutathione transferase-1 by electrospray mass spectrometry.

Microsomal glutathione transferase-1 (MGST1) is a membrane-bound enzyme involved in the detoxification of xenobiotics and the protection of cells against oxidative stress. The proposed active form of the enzyme is a noncovalently associated homotrimer that binds one substrate glutathione molecule/trimer. In this study, this complex has been directly observed by electrospray mass spectrometry analysis of active rat liver MGST1 reconstituted in a minimum amount of detergent. The measured mass of the homotrimer is 53 kDa, allowing for the mass of three MGST molecules in complex with one glutathione molecule. Collision-induced dissociation of the trimer complex resulted in the formation of monomer and homodimer ion species. Two distinct species of homodimer were observed, one unliganded and one identified as a homodimer.glutathione complex. Activation of the enzyme by N-ethylmaleimide through modification of Cys(49) (Svensson, R., Rinaldi, R., Swedmark, S., and Morgenstern, R. (2000) Biochemistry 39, 15144-15149) was monitored by the observation of an appropriate increase in mass in both the denatured monomeric and native trimeric forms of MGST1. Together, the data correspond well with the proposed functional organization of MGST1. These results also represent the first example of direct electrospray mass spectrometry analysis of a detergent-solubilized multimeric membrane protein complex in its native state.

Animals↗
PubMed2003

Basal expression of the human MAPEG members microsomal glutathione transferase 1 and prostaglandin E synthase genes is mediated by Sp1 and Sp3.

Microsomal glutathione transferase (MGST1) and prostaglandin E synthase (PGES) are both members of the MAPEG (Membrane Associated Proteins involved in Eicosanoid and Glutathione metabolism) superfamily. In humans, their organ distribution is quite distinct with the former being widely and constitutively expressed whereas PGES is largely inducible. In order to study the basal expression of these genes, we characterized the promoter regions and identified the elements and the transcription factors required using in vitro assays, including reporter analysis of deletion and mutant clones and EMSA. The results indicate that Sp1 is the protein mediating the basal transcription of MGST1. It appears that both the Sp1 and Sp3 proteins are important for the basal expression of PGES. In addition, mutational analysis of two Barbie-box elements in the PGES promoter showed that these were not involved in the down-regulation of PGES by phenobarbital (PB). These results provide the first description of the basal regulation of these genes in humans.

Base Sequence↗
PubMed2003

Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination.

Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathione-dependent conversion of PGH2 to PGE2 (Vmax; 170 micromol min-1 mg-1) and high kcat/Km (310 mm-1 s-1). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 micromol min-1 mg-1). The formation of 15-hydroperoxy-PGE2 represents an alternative pathway for the synthesis of PGE2, which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 micromol min-1 mg-1), 5-hydroperoxyeicosatetraenoic acid (0.043 micromol min-1 mg-1), and 15-hydroperoxy-PGE2 (0.04 micromol min-1 mg-1). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 micromol min-1 mg-1). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm3/g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1.

Animals↗
PubMed2002

Synthesis and characterization of 6-chloroacetyl-2-dimethylaminonaphthalene as a fluorogenic substrate and a mechanistic probe for glutathione transferases.

Here we demonstrate that the thiol-reactive, environmentally sensitive fluorogenic molecules 6-bromoacetyl-2-dimethylaminonaphthalene and 6-acryloyl-2-dimethylaminonaphthalene are substrates for glutathione transferases (GSTs). Product formation can be measured by strong increase in fluorescence of the glutathione conjugate. As these substances display a high nonenzymatic background reaction rate, we have synthesized and characterized 6-chloroacetyl-2-dimethylaminonaphthalene, which is less reactive, favoring the enzyme-catalyzed reaction. 6-Chloroacetyl-2-dimethylaminonaphthalene was found to be a substrate for all GSTs tested. Apparent k(cat)/K(m) values (ranging between 10 and 500 mM(-1)s(-1)) revealed a strong preference for soluble GSTP1-1, GSTA1-1, and activated MGST1. Thus, 6-chloroacetyl-2-dimethylaminonaphthalene can be used in a highly sensitive assay of these GSTs. 6-Acetyl-2-dimethylaminonaphthalene derivatives are very sensitive toward solvent polarity and potentially also toward properties of binding sites in proteins. Upon binding of the conjugate to GSTs the fluorescence intensity decreased and the emission maximum was blue-shifted. Therefore the interaction of the conjugate with GSTs can be characterized with regard to both binding affinity and kinetics by stopped-flow measurements, and 6-chloroacetyl-2-dimethylaminonaphthalene can be a valuable aid in mechanistic investigations of GSTs, especially those which possess low intrinsic fluorescence.

2-Naphthylamine↗
PubMed2002

Canine sulfotransferase SULT1A1: molecular cloning, expression, and characterization.

Sulfotransferases (SULTs) are involved in detoxification and activation of various endogenous and exogenous compounds including important drugs and hormones. SULT1A, the phenol-SULT subfamily, is the most prominent subfamily in xenobiotic metabolism and has been found in several species, e.g., human, rat, and mouse. We have cloned a phenol-sulfating phenol SULT from dog (cSULT1A1) and expressed it in Escherichia coli for characterization. cSULT1A1 showed 85.8, 82.7, 76.3, and 73.6% identities to human P-PST, human M-PST, rat PST-1, and mouse STp1, respectively. It consists of 295 amino acids, which is in agreement with the human ortholog and sulfate substrates typical for the SULT1A family, i.e., p-nitrophenol (PNP), alpha-naphthol, and dopamine. The K(m) for PNP was found to be within the nanomolar range. It also sulfates minoxidil and beta-estradiol but not dehydroepiandrosterone. Western blot analysis indicated that this newly cloned enzyme was found to be ubiquitously expressed in canine tissues with highest expression in male and female liver.

Amino Acid Sequence↗
PubMed2002

The 3-D structure of microsomal glutathione transferase 1 at 6 A resolution as determined by electron crystallography of p22(1)2(1) crystals.

Pure solubilised microsomal glutathione transferase 1 (MGST1) forms well-ordered two-dimensional (2-D) crystals of two different symmetries, one orthorhombic (p22(1)2(1)) and one hexagonal (p6), both diffracting electrons to a resolution beyond 3 A. A three-dimensional (3-D) map has previously been calculated to 6 A resolution from the hexagonal crystal form. From orthorhombic crystals we have now calculated a 6 A 3-D reconstruction displaying three repeats of four rod-like densities. These are inclined relative to the normal of the membrane plane and consistent with arising from a left-handed four-helix bundle fold. The rendered volume clearly displays the same structural features as the map previously calculated from the p6 crystal type including similar lengths and substructure of the helices, but several distinguishing features do exist. The helices are more tilted in the map calculated from the orthorhombic crystals indicating conformational flexibility. Density present on the cytosolic side is consistent with the location of the active site. In addition, the current map displays the noted similarity to subunit I of cytochrome c oxidase.

Amino Acid Sequence↗
PubMed2002

Repeated blood pressure measurements in a sample of Swedish twins: heritabilities and associations with polymorphisms in the renin-angiotensin-aldosterone system.

BACKGROUND: Twin and family studies have shown that genetic effects explain a relatively high amount of the phenotypic variation in blood pressure. However, many studies have not been able to replicate findings of association between specific polymorphisms and diastolic and systolic blood pressure. METHODS: In a structural equation-modelling framework the authors investigated longitudinal changes in repeated measures of blood pressures in a sample of 298 like-sexed twin pairs from the population-based Swedish Twin Registry. Also examined was the association between blood pressure and polymorphisms in the angiotensin-I converting enzyme and the angiotensin II receptor type 1 with the 'Fulker' test. Both linkage and association were tested simultaneously revealing whether the polymorphism is a Quantitative Trait Locus (QTL) or in linkage disequilibrium with the QTL. RESULTS: Genetic influences explained up to 46% of the phenotypic variance in diastolic and 63% of the phenotypic variance in systolic blood pressure. Genetic influences were stable over time and contributed up to 78% of the phenotypic correlation in both diastolic and systolic blood pressure. Non-shared environmental effects were characterised by time specific influences and little transmission from one time point to the next. There was no significant linkage and association between the polymorphisms and blood pressure. CONCLUSIONS: There is a considerable genetic stability in both diastolic and systolic blood pressure for a 6-year period of time in adult life. Non-shared environmental influences have a small long-term effect. Although associations with the polymorphisms could not be replicated, results should be interpreted with caution due to power considerations.

Aged↗