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R Morgenstern

Publications and source records attributed to R Morgenstern.

At least 55 records · Page 3Linked to original sources
PubMed1999

Common structural features of MAPEG -- a widespread superfamily of membrane associated proteins with highly divergent functions in eicosanoid and glutathione metabolism.

A novel superfamily designated MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), including members of widespread origin with diversified biological functions is defined according to enzymatic activities, sequence motifs, and structural properties. Two of the members are crucial for leukotriene biosynthesis, and three are cytoprotective exhibiting glutathione S-transferase and peroxidase activities. Expression of the most recently recognized member is strongly induced by p53, and may therefore play a role in apoptosis or cancer development. In spite of the different biological functions, all six proteins demonstrate common structural characteristics typical of membrane proteins. In addition, homologues are identified in plants, fungi, and bacteria, demonstrating this superfamily to be generally occurring.

Amino Acid Sequence↗
PubMed1998

A highly active microsomal glutathione transferase from frog (Xenopus laevis) liver that is not activated by N-ethylmaleimide.

Microsomal glutathione transferase has hitherto only been purified from mammalian species. N-ethylmaleimide and trypsin activation (discriminating features of this enzyme) has only been observed in microsomes from mammals. In this paper we describe the first isolation and characterization of a non-mammalian microsomal glutathione transferase from frog (Xenopus laevis) liver. This protein has a molecular weight similar to that of the mammalian enzyme (approximately 17 kDa), but cannot be activated by N-ethylmaleimide or trypsin. In fact the enzyme is rapidly inactivated by this sulfhydryl reagent and protease. It thus appears that N-ethylmaleimide activation is not an obligatory property of microsomal glutathione transferase. The frog liver microsomal glutathione transferase has one of the highest specific activities towards the second substrate 1-chloro-2,4-dinitrobenzene (CDNB) (200 mumol/min mg) obtained with any glutathione transferase and accounts for the high activity found in frog liver microsomes. The kcat/K(m) for glutathione and CDNB are 0.017 and 1.1 x 10(6) M-1 s-1, respectively. The enzyme also functions as a glutathione peroxidase (dilinoleoyl phosphatidylcholine hydroperoxide is reduced (5.2 mumol/min mg)). It is now evident that a highly active microsomal glutathione transferase, with a molecular weight similar to that of the mammalian enzymes also exists in a non-mammal species.

Animals↗
PubMed1998

The mRNA for GST Pi from FRHK rhesus monkey kidney cells codes for an enzyme with activity towards 1-chloro-2,4-dinitrobenzene in spite of an I68F mutation.

A cDNA library was constructed from mRNA of the rhesus monkey kidney cell line, FRHK, and the cDNA sequence for an FRHK glutathione S-transferase (GST) Pi was determined using a RACE method. This represents the first full-length monkey GST Pi sequence to be cloned and determined. The similarity to the human GST Pi was found to be extensive (more than 97%), the deduced protein differing only in six amino acids (aa) positions. FRHK GST Pi was expressed in bacteria and a recombinant protein was purified which demonstrated significant activity towards the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-para-nitrophenoxypropane. Western blots also showed significant amounts of protein, both in the FRHK cells and transformed bacteria. The FRHK GST Pi was found to contain a phenylalanine at aa position 68, a position which is otherwise invariably occupied by an isoleucine in the GST Pi, Alpha, Mu and Beta class enzymes investigated. An isoleucine in this position is thus not essential for activity in the FRHK enzyme, unlike the human GST pi, where the exchange of Ile68 to a tyrosine (Manoharan, T.H, Gulick, A.M., Puchalski, R.B., Servais, A.L., Fahl, W.E., 1992. J. Biol. Chem., 267, 18940-18945), resulted in total loss of activity. Phe68 was mutated to Ile in the FRHK GST Pi enzyme to determine whether the wild type amino acid conferred an impaired catalytic site. The resulting mutant did not show any changes in activity towards CDNB, clearly demonstrating that isoleucine at position 68 is not essential. Thus, the first monkey GST Pi enzyme has been characterized, an enzyme with many similarities to the human forms although it differs in an otherwise conserved residue at aa position 68. This difference does not appear to affect the function of the FRHK GST Pi.

Amino Acid Sequence↗
PubMed1998

Parameters for the two-dimensional crystallization of the membrane protein microsomal glutathione transferase.

Various crystallization parameters were investigated to obtain two-dimensional crystals of the detoxification enzyme microsomal glutathione transferase for structural analysis by electron crystallography. The protein was crystallized by reconstitution of the solubilized trimer into proteoliposomes. Crystallization occurs when minimal amounts of lipid in the range of three lipid molecules per protein trimer are added to the dialysate. Once crystals were obtained, the effect of several parameters on the crystallization was determined. The temperature and initial detergent concentration were found to be crucial parameters in influencing the size of the crystals, and conclusions could be drawn about the rate dependence of the crystallization process. Two highly ordered crystal forms, which are suitable for structural analysis by electron crystallography, were obtained under the two-dimensional crystallization conditions described here.

Animals↗
PubMed1998

Extracellular dopamine in the anterior nucleus accumbens is distinctly affected by ventral tegmental area administration of cholecystokinin and apomorphine: data from in vivo voltammetry.

The interaction of cholecystokinin (CCK) and dopamine (DA) in the mesolimbic system was investigated. The study focused on DAergic cells not containing colocalized CCK projecting from the ventral tegmental area (VTA) to the anterior nucleus accumbens (NA). Differential pulse voltammetry in pargyline pretreated and anesthetized rats was used to measure extracellular DA in the anterior NA following microinjection of apomorphine either alone or in combination with CCK-8s into the VTA. In agreement with an earlier study there was a dose-dependent increase in the DA signal in the anterior NA after microinjection of CCK-8s into the VTA. Apomorphine microinjected into the VTA produced a biphasic effect on extracellular DA in the anterior NA with an increase from basal levels of approximately 50% by 1 ng, whereas 10 ng was ineffective and 100 ng apomorphine caused a slight decrease in the DA signal. Apomorphine (1 ng) microinjected together with 1 ng CCK-8s produced an increase in the DA signal to approximately 180% of the baseline value, whereas the combination of 1 ng apomorphine and 100 ng CCK-8s was ineffective. When 100 ng apomorphine were microinjected in combination with either 1 ng or 100 ng CCK-8s, the DA signal in the anterior NA was unchanged. These results suggest that low doses of apomorphine injected into the VTA synergistically influence the effects of CCK-8s on extracellular DA in the anterior NA, whereas higher doses of apomorphine suppress the effect of CCK-8s on DAergic cells projecting to the anterior NA.

Anesthesia↗
PubMed1998

A simple alternate substrate test can help determine the aqueous or bilayer location of binding sites for hydrophobic ligands/substrates on membrane proteins.

It is of general interest to determine the location of binding sites in membrane protein receptors and enzymes. A simple method is described that relies on the use of alternate substrates with defined hydrophobicity and decipherable trends in relative kcat/Km values. The rates at first-order conditions (low substrate concentrations) for at least two substrates are determined at a defined lipid/detergent volume fraction. Ratios of the rates (which at first-order conditions are linearly related to substrate concentration) are compared to the predicted partitioning of the substrates. Since the relative rates depend also on the kcat/Km ratios, the contribution of this parameter and its limits is discussed. When alternate substrates that fulfill reasonable criteria regarding kcat/Km values are used, the location of binding sites can be tentatively predicted. Examples are given describing evaluation of the location of the xenobiotic binding site in detoxication enzymes (microsomal glutathione transferase and cytochrome P450). Furthermore, it is argued that unspecific binding sites for hydrophobic molecules, such as those that are important in many detoxication enzymes, can only benefit from hydrophobic interactions facing the aqueous compartment of the cell. The membrane location of many drug-metabolizing enzymes thus implies that an aqueous active site located close to the membrane is advantageous, an advantage that could be realized if a concentration gradient of hydrophobic molecules extends into the aqueous phase at lipid interfaces.

Animals↗
PubMed1998

[Reasons for condemnation of slaughtered broilers from two large Swiss producers].

30 broiler flocks from two majors Swiss broiler producing companies were examined for lesions relevant to meat inspection between November 1995 and April 1996. 71.8% of condemned carcasses and viscera were assigned to ascites syndrome, bacterial infections and runting. Other diseases and lesions due to injury or processing were rare. Escherichia coli was responsible for the majority of infections. The average condemnation rate (1.0%) corresponded to findings in literature.

Abattoirs↗
PubMed1998

[Hernia of the lumbar discs in persons at work. Results of a retrospective study in a series of 189 consecutive patients].

INTRODUCTION: Owing to recent advances in the diagnosis and treatment of lumbar disc pathology, the functional results obtained have improved, although there are still certain factors which affect this. The results in the work place are particularly problematic. OBJECTIVE: With the aim of finding an answer to the problem of relapse of the sciatica syndrome in patients operated on for hernia of the lumbar discs, we describe a study carried out in a consecutive group of patients who had had operations for hernias of lumbar discs whilst at work, within the context of revindication involved in this situation. PATIENTS AND METHODS: We describe the results obtained in a series of 189 patients who had been operated on for lumbar disc pathology. Conventional open surgery was performed in 105 cases. Percutaneous nucleotomy was done in the remainder. In 17.39%, operations were done at more than one level, making a total of 265 discs operated on. RESULTS: These were considered good when the patient returned to his former work. Good results were obtained in 72% of the patients treated by open surgery and 68% of those treated by percutaneous nucleotomy. The re-operation rate was 8.57% for open surgery and 32.14% for percutaneous nucleotomy (p < 0.05). CONCLUSIONS: The main reason for re-operation was a recurrence of disc protrusion. Good results were obtained in 80% of those reoperated. All reoperations were done using open surgery techniques. Overall assessment of the cases, including initial and re-operations, gave good results, reaching 83.59% in the current series.

Adult↗
PubMed1997

The 3.0 A projection structure of microsomal glutathione transferase as determined by electron crystallography of p 21212 two-dimensional crystals.

Two-dimensional crystals of rat microsomal glutathione transferase were grown during dialysis of detergent-solubilized enzyme after addition of a small amount of phospholipid. The crystals had two-sided plane group symmetry p21212 with a calibrated unit cell size of a=91.90 A, b=90.83 A. Electron diffraction patterns were recorded showing significant reflections extending to 3.0 A. A combination of these structure factor amplitudes with phases from high-resolution images following image processing was used to calculate a projection map of the protein. The asymmetric unit of the structure consists of three microsomal glutathione transferase molecules. The local 3-fold axis at the center of the trimer is delineated by six parallel alpha-helices, two from each monomer. The two helices differ significantly in their respective projection structure. The inner helical core of the trimer is partly surrounded by elongated domains with extensions towards the helices and which contain resolved density maxima at a spacing of 4 to 5 A. A well-defined strong peak is localized close to the elongated domain and at a distance of about 9.5 A from two of the inner helices.

Crystallization↗
PubMed1997

Binding of glutathione and an inhibitor to microsomal glutathione transferase.

Microsomal glutathione transferase is an abundant liver protein that can be activated by thiol reagents. It is not known whether the activation is associated with changed binding properties of the enzyme. Therefore the binding of GSH and an inhibitor to rat liver microsomal glutathione transferase was studied by use of equilibrium dialysis and equilibrium partition in a two-phase system. The radioactive substrate glutathione and an inhibitor (glutathione sulphonate) give hyperbolic binding isotherms with a stoichiometry of 1 mol per mol of enzyme (i.e. 1 molecule per homotrimer). Glutathione had an equilibrium binding constant of 18 microM. Competition experiments involving glutathione sulphonate showed that it could effectively displace GSH. These and kinetic studies showed that the Kd and Ki for glutathione sulphonic acid are close to 10 microM. No change in these parameters was obtained after N-ethylmaleimide activation of the enzyme. Thus activation does not result from changes in binding affinity to GSH.

Animals↗
PubMed1997

Effects of peroxisome proliferators and/or hypothyroidism on xenobiotic-metabolizing enzymes in rat testis.

The objectives of the present work were to study the effects of certain peroxisome proliferators on xenobiotic-metabolizing enzyme activities in the testes of normal and hypothyroid rats, i.e. phenol sulfotransferases (pST), phenol UDP-glucuronosyl transferases (pUDPGT), glutathione transferases (GST), catalase, epoxide hydrolase (EH), glutathione peroxidase (GPX) and NAD(P)H quinone oxidoreductase (QR). Adult male rats (normal and hypothyroid) were treated for 10 days with clofibrate (0.5%), perfluorooctanoic acid (0.5%, PFOA), acetylsalisylic acid (1%, ASA) and di(2-ethylhexyl)phthalate (2%, DEHP) in their diet. The results show that treatment of normal rats with peroxisome proliferators dramatically affects the activities of xenobiotic-metabolizing enzymes (40-60% reduction). The highest effects are seen in catalase activity (50-60% with PFOA and ASA), pUDPGT (55% with PFOA), pST (55% with PFOA) and QR (50% with DEHP). These effects are not seen or are weaker after induction of hypothyroidism. Taken together, it is concluded that different classes of peroxisome proliferators have different effects on rat testicular xenobiotic-metabolizing enzymes.

Administration, Oral↗
PubMed1997

Structural and functional aspects of rat microsomal glutathione transferase. The roles of cysteine 49, arginine 107, lysine 67, histidine, and tyrosine residues.

Rat liver microsomal glutathione transferase is rapidly inactivated upon treatment with the arginine-selective reagent phenylglyoxal or the lysine-selective 1,3,5-trinitrobenzenesulfonate. Glutathione sulfonate, an inhibitor of the enzyme, gives nearly complete protection against inactivation and prevents modification, indicating that these residues form part of or reside close to the active site. Sequence analysis of peptides from peptic and tryptic digests of [7-14C]phenylglyoxal- and 1,3,5-trinitrobenzenesulfonate-treated microsomal glutathione transferase indicated arginine 107 and lysine 67 as the sites of modification. A set of mutant forms of microsomal glutathione transferase was constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli BL21(DE3). Arginine 107 was exchanged for alanine and lysine residues. The alanine mutant (R107A) exhibited an activity and inhibition profile similar to that of the wild type enzyme but displayed a decreased thermostability. Thus, arginine 107 does not appear to participate in catalysis or substrate binding; instead, an important structural role is suggested for this residue. Lysine 67 was mutated to alanine and arginine with no effect on activity. All three histidines were replaced by glutamine, and the resulting mutant proteins had activities comparable with that of the wild type. It can thus be concluded that the chemical modification experiments indicating that arginine 107, lysine 67, and one of the histidines partake in catalysis can be disproved. However, protection from modification by a competitive inhibitor indicates that these residues could be close to the glutathione binding site. All tyrosine to phenylalanine substitutions resulted in mutants with activities similar to that of the wild type. Interestingly, the exchange of tyrosine 137 appears to result in activation of the enzyme. Thus, the microsomal glutathione transferase must display an alternate stabilization of the thiolate anion of glutathione other than through interaction with the phenolic hydroxyl group of a tyrosine residue. Substitution of cysteine 49 with alanine resulted in a semiactivated mutant enzyme with enzymatic properties partly resembling the activated form of microsomal glutathione transferase. The function of this mutant was not altered upon reaction with N-ethylmaleimide, and cysteine 49 is thus demonstrated as the site of modification that results in activation of microsomal glutathione transferase.

Amino Acid Sequence↗
PubMed1997

Glutathione transferase mimics: micellar catalysis of an enzymic reaction.

Substances that mimic the enzyme action of glutathione transferases (which serve in detoxification) are described. These micellar catalysts enhance the reaction rate between thiols and activated halogenated nitroarenes as well as alpha,beta-unsaturated carbonyls. The nucleophilic aromatic substitution reaction is enhanced by the following surfactants in descending order: poly(dimethyldiallylammonium - co - dodecylmethyldiallylammonium) bromide (86/14) >>cetyltrimethylammonium bromide>zwittergent 3-16 (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate)>zwittergent+ ++ 3-14 (n-tetradecyl-N,N-dimethyl - 3 - ammonio -1 - propanesulphonate) approximately N,N - dimethyl - laurylamine N-oxide>N,N-dimethyloctylamine N-oxide. The most efficient catalyst studied is a polymeric material that incorporates surfactant properties (n-dodecylmethyldiallylammonium bromide) and opens up possibilities for engineering sequences of reactions on a polymeric support. Michael addition to alpha,beta-unsaturated carbonyls is exemplified by a model substance, trans-4-phenylbut-3-en-2-one, and a toxic compound that is formed during oxidative stress, 4-hydroxy-2-undecenal. The latter compound is conjugated with the highest efficiency of those tested. Micellar catalysts can thus be viewed as simple models for the glutathione transferases highlighting the influence of a positive electrostatic field and a non-specific hydrophobic binding site, pertaining to two catalytic aspects, namely thiolate anion stabilization and solvent shielding.

Animals↗
PubMed1997

Cholecystokinin increases extracellular dopamine overflow in the anterior nucleus accumbens via CCK(B) receptors in the VTA assessed by in vivo voltammetry.

Differential pulse voltammetry was used to investigate the extracellular dopamine (DA) and DOPAC signal in the anterior part of nucleus accumbens (N.acc.) after microinjection of cholecystokinin (CCK) derivatives into the ventral tegmental area (VTA). Both the mixed CCK(A)/CCK(B) receptor agonist CCK-8s and the selective CCK(B) receptor agonist CCK-4 caused a dose-dependent increase in the DA signal after doses of 10 ng and 100 ng while CCK-8s had no effect on the DOPAC signal. The CCK(A) receptor antagonist L 364,718 (25 microg/kg i.p.) as well as the CCK(B) receptor antagonist L 365,260 (25 microg/kg i.p.) were administered prior to microinjection of 100 ng CCK-8s and L 365,260, but not L 364,718, completely inhibiting the DA increase produced by CCK-8s. Analysis of the tissue levels of DA and its main metabolites in the anterior part of N.acc. revealed no changes after CCK-8s microapplication into VTA. The presented data indicate a CCK(B) receptor-mediated increase in extracellular DA in the anterior N.acc. after microapplication of CCK derivatives into the VTA.

Animals↗
PubMed1997

Unilateral fetal mesencephalic grafts in intact rats reduce amphetamine-induced dopamine release in both striata. An in vivo voltammetric study.

This study investigated amphetamine-induced striatal dopamine release after intraventricular unilateral fetal mesencephalic grafts in otherwise intact rats. Dopamine was monitored in vivo by differential pulse voltammetry. In grafted animals, amphetamine-induced dopamine release was decreased compared to sham-grafted, age-matched controls. This decrease was observed in the grafted as well as in the contralateral striatum five months after intraventricular grafting. There was no measurable effect of the graft on the amphetamine-induced rotational behaviour. Our results exceed former observations reporting decreased amphetamine-induced dopamine release in the contralateral striatum of 6-hydroxydopamine-lesioned and unilaterally-grafted rats which had been attributed to a reduction of dopamine transporters. Furthermore, it was shown that concerning this effect ventral mesencephalic grafts are independent of a previous 6-hydroxydopamine lesion.

Amphetamine↗
PubMed1996

Interaction of human leukotriene C4 synthase and microsomal glutathione transferase in vivo.

Microsomal glutathione transferase (mGT) specifically binds leukotriene C4 synthase in the presence of Mg2+ ion (Söderström et al., Protein Expression and Purification (1995) 6, 352-356). To investigate if this interaction occurs in vivo we screened a human lung cDNA library with a bait vector encoding human mGT in the yeast two-hybrid system. One of the five positive clones obtained encoded leukotriene C4 synthase. This clone was expressed in two heterologous systems. The recombinant protein cross-reacted with a guinea pig antibody raised against a Keyhole limpet hemocyanin coupled synthetic peptide corresponding to amino acids 141-150 of human leukotriene C4 synthase.

Amino Acid Sequence↗
PubMed1996

Membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli.

Rat liver microsomal glutathione transferase is a mammalian membrane protein that can be successfully expressed in Escherichia coli in an enzymatically active form. The protein does not form inclusion bodies and is recovered in the membrane fraction. The membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli was investigated by comparing the proteolytic cleavage products from intact and permeabilized spheroplasts. It was shown that lysine-4 of microsomal glutathione transferase is directed towards the outside, whereas lysine-41 faces the inside of the E. coli inner membrane. This shows that microsomal glutathione transferase has an inside-out orientation in E. coli spheroplasts as compared to liver microsomes. This fact enables us to make topology experiments that were previously not possible. Intact spheroplasts treated with pronase yielded a cleavage pattern consistent with two additional exposed segments closer to the C-terminus. Thus a polytopic model is suggested for the membrane association of microsomal glutathione transferase.

Animals↗