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M Löffler

Publications and source records attributed to M Löffler.

At least 37 records · Page 2Linked to original sources
PubMed2000

Role of endogenous nitric oxide in circadian blood pressure regulation in healthy humans and in patients with hypertension or atherosclerosis.

BACKGROUND: Nitric oxide (NO) is involved in the regulation of blood pressure and local blood flow. Its biological activity is impaired in hypertension and atherosclerosis. Because blood pressure undergoes a circadian rhythm, we investigated whether systemic NO production is dependent on a circadian variability, and whether the phasing of diurnal rhythm in NO production corresponds to the one in blood pressure in humans. METHODS: We studied three groups of human subjects: 8 healthy volunteers (HV), 8 patients with essential hypertension (HT), and 8 patients with peripheral arterial occlusive disease (PAOD). Twenty-four-hour ambulatory blood pressure monitoring was performed simultaneously with eight consecutive 3-hour urine collection periods. Urinary nitrate excretion was measured by gas chromatography-mass spectrometry; urinary cyclic GMP excretion was assessed by RIA. RESULTS: Twenty-four-hour mean arterial blood pressure was 119.8 +/- 2.0/75.8 +/- 1.5 mm Hg in HV, 145.0 +/- 6.4/94.9 +/- 2.8 mm Hg in HT (P < 0.05 vs HV), and 137.0 +/- 7.3/81.5 +/- 1.9 mm Hg in PAOD (P = NS vs HV). There was significant circadian variation in blood pressure in all groups, but daily amplitude was lower in HT and PAOD than in HV (P < 0.05); 24-hour mean urinary nitrate excretion was 183.4 +/- 27.2 mumol/mmol creatinine in HV, 102.9 +/- 18.1 mumol/mmol creatinine in HT, and 162.1 +/- 22.2 mumol/mmol creatinine in PAOD (P < 0.05 vs HV and HT). Urinary cyclic GMP excretion was 211.8 +/- 19.0 nmol/mmol creatinine in HV, 108.6 +/- 12.4 nmol/mmol creatinine in HT, and 97.9 +/- 13.4 nmol/mmol creatinine in PAOD (P < 0.05 for HT and PAOD vs HV). Circadian variation was present in urinary nitrate and cyclic GMP excretion in HV but was significantly diminished in HT and PAOD, respectively; 24-hour mean nitrate-to-cyclic GMP ratio was 0.89 +/- 0.05 in HV and 1.10 +/- 0.10 in HT (P = NS). It was increased to 2.02 +/- 0.17 in PAOD (P < 0.05 vs HV and HT). CONCLUSIONS: There is significant circadian variation in urinary nitrate and cyclic GMP excretion rates, two marker molecules for systemic NO production, in healthy humans. NO production is increased in the morning, concomitantly with the morning increase in blood pressure, indicating that NO may buffer blood pressure increase. Diurnal variation in nitrate and cyclic GMP excretion is absent in HT, pointing to impaired NO formation. The major change in PAOD is increased nitrate/cyclic GMP ratio, which points to increased oxidative inactivation of NO in this disease. Disturbed formation and activity of NO may contribute to blood pressure alterations in cardiovascular disease.

Adult↗
PubMed1999

c-Myc does not prevent glucocorticoid-induced apoptosis of human leukemic lymphoblasts.

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. The signal transduction pathways leading to this clinically-relevant form of apoptosis have, however, not been sufficiently elucidated. GC bind to their specific receptor, a ligand-activated transcription factor of the Zn-finger type, that activates or represses transcription of GC-responsive genes. Previous studies in leukemia cells suggested that transcriptional repression of c-myc expression might be the crucial event in GC-induced apoptosis, although in other systems, c-Myc apparently increased the sensitivity to cell-death inducers. To address this controversy, we stably transfected the GC-sensitive human T-ALL cell line CEM-C7H2 with constructs allowing tetracycline-regulated expression of c-Myc. Subsequent analyses of these cell lines showed that overexpression of c-Myc per se had little, if any, effect on cell viability, although it rendered the cells more sensitive to apoptosis induced by low serum, confirming the functionality of the expressed transgene. More importantly, however, when the cells were treated with GC in the presence of exogenous c-Myc, they underwent apoptosis exceeding that in cells treated in the absence of transgenic c-Myc. The data indicate that c-myc downregulation is not critical for induction of cell-death by GC in this system, and support the notion that c-Myc sensitizes cells to apoptosis-inducing agents.

Apoptosis↗
PubMed1999

Bcl-2 interferes with the execution phase, but not upstream events, in glucocorticoid-induced leukemia apoptosis.

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. Cell death is associated with activation of members of the interleukin-1beta-converting enzyme (ICE) protease/caspase family and, is presumably prevented by the anti-apoptotic protein Bcl-2. To further address the role of Bcl-2 in GC-mediated cytotoxicity, we generated subclones of the GC-sensitive human T-cell acute lymphoblastic leukemia line CCRF-CEM, in which transgenic Bcl-2 expression is regulated by tetracycline. Up to about 48 h, exogenous Bcl-2 almost completely protected these cells from apoptosis, digestion of poly-ADP ribose polymerase (PARP) and generation of Asp-Glu-Val-Asp cleaving (DEVDase) activity. However, when the cells were cultured for another 24 h in the continuous presence of GC, they underwent massive apoptosis that was associated with DEVDase activity and PARP cleavage. Bcl-2 did not markedly affect GC-mediated growth arrest, thereby separating the anti-proliferative from the apoptosis-inducing effect of GC. Moreover, Bcl-2 did not prevent the dramatic reduction in the levels of several mRNAs observed during GC treatment, including the transgenic Bcl-2 mRNA. Thus, Bcl-2 can be placed upstream of effector caspase activation, but downstream of other GC-regulated events, such as growth arrest and the potentially critical repression of steady state levels of multiple mRNA.

Apoptosis↗
PubMed1999

GRC5 and NMD3 function in translational control of gene expression and interact genetically.

The yeast gene, GRC5 (growth control), is a member of the highly conserved QM gene family, the human member of which has been associated with the suppression of Wilms' tumor. GRC5 encodes ribosomal protein L10, which is thought to play a regulatory role in the translational control of gene expression. A revertant screen identified four spontaneous revertants of the mutant grc5-1ts allele. Genetic and phenotypic analysis showed that these represent one gene, NMD3, and that the interaction of NMD3 and GRC5 is gene-specific. NMD3 was previously identified as a component of the nonsense-mediated mRNA decay pathway. The point mutations within NMD3 reported here may define a domain important for the functional interaction of Grc5p and Nmd3p.

Alleles↗
PubMed1999

Interaction between dexamethasone and butyrate in apoptosis induction: non-additive in thymocytes and synergistic in a T cell-derived leukemia cell line.

In thymocytes butyrate and trichostatin A are unable to augment dexamethasone-induced apoptosis. In cultured rat thymocytes the extent of apoptosis induced by dexamethasone alone did not increase by addition of 0.1 - 10 mM butyrate. Even more pronounced was the non-additive interrelationship between dexamethasone and trichostatin A, as trichostatin A-induced apoptosis was not only blocked by the presence of dexamethasone but dexamethasone-induced apoptosis was also partially inhibited in the presence of 0.1 - 0.5 microM trichostatin A. The fact that the non-additive relationship with dexamethasone for apoptosis induction was observed with both histone deacetylase inhibitors suggests that in thymocytes this phenomenon is related to histone acetylation. In contrast to this, in the human T cell-derived leukemia cell line CEM-C7H2, dexamethasone did not block butyrate- or trichostatin A-induced apoptosis; moreover, butyrate, in the concentration range of 0.1 - 1 mM, had a marked synergistic effect on dexamethasone-induced apoptosis. This synergism, however, was not mimicked by trichostatin A, indicating that the effect is not related to histone acetylation but rather due to a pleiotropic effect of butyrate. Furthermore, in CEM-C7H2 cells, at higher concentrations of butyrate (5 - 10 mM) or trichostatin A (0.4 - 0.8 microM), there was a minor but reproducible antagonistic effect of dexamethasone on apoptosis induced by each of the two histone deacetylase inhibitors, suggesting that this antagonistic effect too, is related to histone hyperacetylation.

Animals↗
PubMed1999

Apoptosis induced by the histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts.

The histone deacetylase inhibitor and potential anti-cancer drug sodium butyrate is a general inducer of growth arrest, differentiation, and in certain cell types, apoptosis. In human CCRF-CEM, acute T lymphoblastic leukemia cells, butyrate, and other histone deacetylase inhibitors caused G2/M cell cycle arrest as well as apoptotic cell death. Forced G0/G1 arrest by tetracycline-regulated expression of transgenic p16/INK4A protected the cells from butyrate-induced cell death without affecting the extent of histone hyperacetylation, suggesting that the latter may be necessary, but not sufficient, for cell death induction. Nuclear apoptosis, but not G2/M arrest, was delayed but not prevented by the tripeptide broad-range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD) and, to a lesser extent, by the tetrapeptide 'effector caspase' inhibitors benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Glu-Ile-Asp.fluoromethyl-ketone (VEID); however, the viral protein inhibitor of 'inducer caspases', crmA, had no effect. Bcl-2 overexpression partially protected stably transfected CCRF-CEM sublines from butyrate-induced apoptosis, but showed no effect on butyrate-induced growth inhibition, further distinguishing these two butyrate effects. c-myc, constitutively expressed in CCRF-CEM cells, was down-regulated by butyrate, but this was not causative for cell death. On the contrary, tetracycline-induced transgenic c-myc sensitized stably transfected CCRF-CEM derivatives to butyrate-induced cell death.

Apoptosis↗
PubMed1999

L-arginine stimulates NO-dependent vasodilation in healthy humans--effect of somatostatin pretreatment.

BACKGROUND: L-arginine is the precursor of endogenous nitric oxide (NO) and a potent stimulator of pituitary growth hormone and pancreatic insulin secretion. Both hormones have vasodilatory effects, which may be mediated via NO. We investigated whether growth hormone and/or insulin secretion contributes to L-arginine-induced vasodilation. METHODS: Ten healthy male subjects received an intravenous infusion of L-arginine, or L-arginine during somatostatin coinfusion lasting 30 minutes. Blood pressure, heart rates, and total peripheral resistance were assessed from 30 minutes prior to L-arginine infusion to 120 minutes after the start of the infusion. Plasma nitrite and cGMP concentrations were used as markers for endogenous NO formation. RESULTS: L-arginine significantly reduced total peripheral resistance, which remained decreased for 60 minutes after the end of the infusion. This resulted in a significantly lowered blood pressure. L-arginine elevated plasma nitrite and cGMP concentrations. Plasma growth hormone level showed a peak at 30 minutes after the infusion, while insulin and glucagon levels were maximal during the infusion, these endocrine effects were blocked during somatostatin coinfusion. The initial reduction in total peripheral resistance and blood pressure, and the elevation of nitrite and cGMP levels were still present during somatostatin cotreatment, but values returned to baseline more rapidly at the end of the L-arginine infusion. CONCLUSION: We conclude that growth hormone contributes to the late phase of L-arginine-induced, NO-mediated vasodilation. By contrast, insulin did not mediate L-arginine induced vasodilation, as the early vasodilator effect, which occurred concomitantly with the peak insulin secretion, was still present after insulin secretion was blocked with somatostatin.

Adult↗
PubMed1998

Species-related inhibition of human and rat dihydroorotate dehydrogenase by immunosuppressive isoxazol and cinchoninic acid derivatives.

The isoxazol leflunomide (N-(4-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide) and its active metabolite A77-1726 (N-(4-trifluoromethyl)-phenyl-2-cyano-3-hydroxy-crotonic acidamide) are promising disease-modifying antirheumatic drugs now in clinical trials. The malononitrilamides MNA279 (2-cyano-3-cyclopropyl-3-oxo-(4-cyanophenyl)propionamide) MNA715(N-(4-trifluoromethyl)-phenyl-2-cyano-3-hydroxy-hept-2-en-6- in-carboxylic acidamide) and HR325 (1(3-methyl-4-trifluoro methylphenyl-carbamoyl)-2-cyclopropyl-2oxo-propionitrile) were shown to block rejection after allograft and xenograft transplantation in animals. Brequinar and other cinchoninic acid derivatives have also been evaluated as immuno-suppressive agents. A77-1726, HR325 and brequinar have been shown to have strong inhibitory effects on mitochondrial dihydroorotate dehydrogenase [EC 1.3.99.11], the fourth enzyme of pyrimidine de novo synthesis, with concomitant reduction of pyrimidine nucleotide pools. Pyrimidine nucleotides are essential for normal immune cell functions. Because most investigations had been carried out with cells, cell homogenates or mitochondrial fractions, it was the rationale of the present study to differentiate, under standardized conditions, the effect of leflunomide, A77-1726, MNA279, MNA715, HR 325 and brequinar on the recombinant rat and human enzymes, which were purified in our laboratory. Whereas leflunomide was a relatively weak inhibitor of the rat (IC50 = 6.3 microM) and human (IC50 = 98 microM) dihydroorotate dehydrogenase, the influence of A77-1726, MNA 279, MNA715 and HR325 was of comparable efficacy for either the rat (range of IC50, 19-53 nM) or the human enzyme (range of IC50, 0.5-2.3 microM). From the IC50 values, it was deduced that brequinar was a more potent inhibitor of the human dihydroorotate dehydrogenase activity (IC50 = 10 nM) than of the rat enzyme (IC50 = 367 nM). The rat enzyme was influenced by all isoxazol derivatives to a greater extent (IC50 = 19 nM A77-1726) than the human enzyme (IC50 = 1.1 microM A77-1726). These results may provide a plausible explanation for the findings of other laboratories with cultured cell lines and lymphocytes: in comparison to cells derived from human tissues, rat and other rodent cells were more susceptible to the isoxazol derivatives and less susceptible to brequinar. Our detailed kinetic investigations of the bisubstrate reaction catalyzed by rat dihydroorotate dehydrogenase revealed a noncompetitive type of inhibition by A77-1726 with respect to the substrate dihydroorotate and the cosubstrates ubiquinone or decylubiquinone. For brequinar, the inhibition was noncompetitive with respect to the substrate dihydroorotate, whereas with the quinone it was found to follow the "mixed typed" inhibition. In addition, brequinar acted as a "slow-binding" inhibitor of the human dihydroorotate dehydrogenase, a feature that might be of consequence for the reversibility of the reaction with the target.

Androstenols↗
PubMed1998

Structural and functional comparison of agents interfering with dihydroorotate, succinate and NADH oxidation of rat liver mitochondria.

Mitochondrially bound dihydroorotate dehydrogenase (EC 1.3.99.11) catalyses the fourth sequential step in the de novo synthesis of uridine monophosphate; this enzyme uses ubiquinone as the proximal and cytochrome oxidase as is the ultimate electron transfer system. Here, seven compounds with proven antiproliferative activity and in vitro antipyrimidine effects were investigated with isolated functional mitochondria of rat tissues in order to differentiate their anti-dihydroorotate dehydrogenase potency versus putative effects on the respiratory chain enzymes. Ten microM of brequinar sodium, the leflunomide derivatives A77-1726, [2-cyano-3-cyclopropyl-3-hydroxy-enoic acid (4-trifluoromethylphenyl)-amide], MNA 279, (2-cyano-N-(4-cyanophenyl)-3-cyclopropyl-3-oxo-propanamide), MNA715 (2-cyano-3-hydroxy-N-(4-(trifluoromethyl)-phenyl-6-heptanamide), HR325 (2-cyano-3-cyclopropyl-3-hydroxy-N-[3'-methyl-4'-(trifluoromethyl)phenyl ]-propenamide), and the diazine toltrazuril completely inhibited the dihydroorotate-induced oxygen consumption of liver mitochondria. Succinate and NADH oxidation were found to be influenced only at elevated drug concentration (100 microM), with the exception of HR325, 10 microM of which caused a 70% inhibition of NADH and 50% inhibition of succinate oxidation. This was comparable to the effects of toltrazuril, which caused an approximate 75% inhibition of NADH oxidation. Ciprofloxacin was shown here to have only marginal effects on the redox activities of the inner mitochondrial membrane. This differentiation of drug effects on mitochondrial functions will contribute to a better understanding of the in vivo pharmacological activity of these drugs, which are presently in clinical trials because of their immunosuppressive, cytostatic or anti-parasitic activity. A comparison of the influence of A77-1726, HR325, brequinar and 2,4-dinitrophenol on energetically coupled rat liver mitochondria revealed only a weak uncoupling potential of A77-1726 and brequinar. In addition, a modeling study was raised to search for common spatial arrangements of functional groups essential for binding of inhibitors to dihydroorotate dehydrogenase. From the structural comparison of different metabolites and inhibitors of pyrimidine metabolism, a 6-point model was obtained by conformational analysis for the drugs tested on mitochondrial functions, pharmacophoric perception and mapping. We propose our model in combination with kinetic data for a rational design of highly specific inhibitors of dihydroorotate dehydrogenase.

Aniline Compounds↗
PubMed1998

Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase.

Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system in Trichoplusia ni cells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight histidines. Extracts from mitochondria of Spodoptera frugiperda cells infected with the recombinant virus using Triton X-100 were loaded onto Ni2+-nitrilotriacetic acid agarose and histidine-tagged rat protein was selectively eluted with imidazole-containing buffer. In view of our previously published work, the quality of the electrophoretic homogenous rat enzyme was markedly improved; specific activity was 130-150 micromol dihydroorotate/min per milligram; and the stoichiometry of flavin content was 0.8-1.1 mol/mol protein. Efforts to generate mammalian dihydroorotate dehydrogenases with low production costs from bacteria resulted in successful overexpression of the carboxy-terminal-modified rat and human dihydroorotate dehydrogenase in XL-1 Blue cells. By employing the metal chelate affinity chromatography under native conditions, the histidine-tagged human enzyme was purified with a specific activity of 150 micromol/min/mg and the rat enzyme with 83 micromol/min/mg, respectively, at pH 8.0-8.1 optimum. Kinetic constants of the recombinant histidine-tagged rat enzyme from bacteria (dihydroorotate, Km = 14.6 micromol electron acceptor decylubiquinone, Km = 9.5 micromol) were close to those reported for the enzyme from insect cells, with or without the affinity tag. HPLC analyses identified flavin mononucleotide as cofactor of the rat enzyme; UV-vis and fluorometric analyses verified a flavin/protein ratio of 0.8-1.1 mol/mol. By spectral analyses of the functional flavin with the native human enzyme, the interaction of the pharmacological inhibitors Leflunomide and Brequinar with their target could be clarified as interference with the transfer of electrons from the flavin to the quinone. The combination of the bacterial expression system and metal chelate affinity chomatography offers an improved means to purify large quantities of mammalian membrane-bound dihydroorotate dehydrogenases which, by several criteria, possesses the same functional activities as non-histidine-tagged recombinant enzymes.

Animals↗
PubMed1997

p53-induced apoptosis in the human T-ALL cell line CCRF-CEM.

The tumor suppressor p53 has been implicated in apoptosis induction and is mutated in human T-ALL CCRF-CEM cells. To investigate possible consequences of wild-type p53 loss, we reconstituted CEM-C7H2, a subclone of CCRF-CEM, with a temperature-sensitive p53 allele (p53ts). Stably transfected lines expressed high levels of p53ts and shift to the permissive temperature (32 degrees C) caused rapid induction of p53-regulated genes, such as p21(CIP1/WAF1), mdm-2 and bax. This was followed by extensive apoptosis within 24 h to 36 h, supporting the notion that mutational p53 inactivation contributed to the malignant phenotype. p53-dependent apoptosis was preceded by digestion of poly(ADP-ribose) polymerase, a typical target of interleukin-1beta-converting enzyme (ICE)-like proteases/caspases, and was markedly resistant to the ICE/caspase-1 and FLICE/caspase-8 inhibitor acetyl-Tyr-Val-Ala-Asp.chloromethylketone (YVAD), but sensitive to the CPP32/caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD), a caspase inhibitor with broader specificity. This indicated an essential involvement of caspases, but argued against a significant role of ICE/caspase-1 or FLICE/caspase-8. Actinomycin D or cycloheximide prevented cell death, suggesting that, in this system, p53-induced apoptosis depends upon macromolecule biosynthesis. Introduction of functional p53 into CEM cells enhanced their sensitivity to the DNA-damaging agent doxorubicin, but not to the tubulin-active compound vincristine. Thus, mutational p53 inactivation in ALL might entail relative resistance to DNA-damaging, but not to tubulin-destabilizing, chemotherapy.

Alleles↗
PubMed1997

Rat dihydroorotate dehydrogenase: isolation of the recombinant enzyme from mitochondria of insect cells.

Mammalian dihydroorotate dehydrogenase (EC 1.3.99.11), the fourth enzyme of pyrimidine de novo synthesis is located in the mitochondrial inner membrane with functional connection to the respiratory chain. From the cDNA of rat liver dihydroorotate dehydrogenase cloned in our laboratory the first complete sequence of a mammalian enzyme was deduced. Two hydrophobic stretches centered around residues 20 and 357, respectively, and a short N-terminal mitochondrial targeting sequence of 10 amino acids was proposed. A recombinant baculovirus containing the rat liver cDNA for dihydroorotate dehydrogenase was constructed and used for virus infection and protein expression in Trichoplusia ni cells. The targeting of the recombinant protein to mitochondria of the insect cells was monitored by activity determination of dihydroorotate dehydrogenase in subcellular compartments in comparison to succinate dehydrogenase activity (EC 1.3.5.1), which is a specific marker enzyme of the inner mitochondrial membrane. The results of subcellular distribution were verified by Western blotting with anti-dihydroorotate dehydrogenase immunoglobulins. The activity of the recombinant enzyme in the mitochondria of infected insect cells was found to be about 570-fold above the level of dihydroorotate dehydrogenase in rat liver mitochondria. By cation exchange chromatography of the Triton X-114 solubilisate of mitochondria, dihydroorotate dehydrogenase was purified to give a specific activity of 15 U/mg at pH 8.0. This was a marked progress over the six-step purification procedure of the enzyme from rat liver which resulted in a specific activity of 0.7 U/mg at pH 8.0. The characteristic flavin absorption spectrum obtained with the recombinant enzyme gave strong evidence that the rodent enzyme is a flavoprotein. By enzyme kinetic studies K(m) values for dihydroorotate and ubiquinone were 6.4 and 9.9 microM with the recombinant enzyme, and were 5.0 and 19.7 microM, respectively, with the rat liver enzyme. After expression of only truncated forms of human dihydroorotate dehydrogenase, the present successful generation of the complete rodent enzyme using insect cells and the efficient procedure will promote structure and function studies of the eukaryotic dihydroorotate dehydrogenases in comparison to the microbial enzyme.

Amino Acid Sequence↗
PubMed1997

Dihydroorotat-ubiquinone oxidoreductase links mitochondria in the biosynthesis of pyrimidine nucleotides.

Pyrimidines and purine (deoxy)nucleotides are the building blocks of DNA and RNA. Nucleoside diphosphate sugars, e.g. UDP-glucose, are the reactive intermediates in the synthesis of nearly all glycosidic bonds between sugars. In mammals the requirement for pyrimidines is met by UMP de novo synthesis and, to a greater or lesser extent, by salvage of free nucleosides. The exceptional compartmentation of the de novo synthesis with respect to mitochondrially-bound dihydroorotate dehydrogenase ('DHOdehase' or 'DHODH', EC 1.3.99.11) is one focus of the present work. DHODH activity was determined by the dihydroorotate-dependent oxygen consumption or by the UV absorption of the product orotate with mitochondria isolated from rodent and porcine tissues. For comparison, the cytochrome c and choline-dependent oxygen consumption of mitochondria from different tissues was measured. The highest specific activity of the rat DHODH was found in liver (2.3 x 10(-3) mumol/min x mg protein) > kidney > heart. The application of known enzyme inhibitors Brequinar Sodium and Leflunomide for DHODH and sodium cyanide for cytochrome c oxidase verified the specificity of the activity tests used. The relation of DHODH activity versus that of cytochrome c oxidase revealed the lowest ratios in heart mitochondria and the highest in liver mitochondria. Since disorders in the mitochondrial energy metabolism could entail severe impairment of pyrimidine biosynthesis via respiratory-chain coupled DHODH, it is suggested to include improvement of pyrimidine nucleotide status in therapy protocols.

Animals↗
PubMed1996

Functional expression of a fragment of human dihydroorotate dehydrogenase by means of the baculovirus expression vector system, and kinetic investigation of the purified recombinant enzyme.

Human mitochondrial dihydroorotate dehydrogenase (the fourth enzyme of pyrimidine de novo synthesis) has been overproduced by means of a recombinant baculovirus that contained the human cDNA fragment for this protein. After virus infection and protein expression in Trichoplusia ni cells (BTI-Tn-5B1-4), the subcellular distribution of the recombinant dihydroorotate dehydrogenase was determined by two distinct enzyme-activity assays and by Western blot analysis with anti-(dihydroorotate dehydrogenase) Ig. The targeting of the recombinant protein to the mitochondria of the insect cells was verified. The activity of the recombinant enzyme in the mitochondria of infected cells was about 740-fold above the level of dihydroorotate dehydrogenase in human liver mitochondria. In a three-step procedure, dihydroorotate dehydrogenase was purified to a specific activity of greater than 50 U/mg. Size-exclusion chromatography showed a molecular mass of 42 kDa and confirmed the existence of the fully active enzyme as a monomeric species. Fluorimetric cofactor analysis revealed the presence of FMN in recombinant dihydroorotate dehydrogenase. By kinetics analysis, Km values for dihydroorotate and ubiquinone-50 were found to be 4 microM and 9.9 microM, respectively, while Km values for dihydroorotate and decylubiquinone were 9.4 microM and 13.7 microM, respectively. The applied expression system will allow preparation of large quantities of the enzyme for structure and function studies. Purified recombinant human dihytdroorotate dehydrogenase was tested for its sensitivity to a reported inhibitor A77 1726 (2-hydroxyethyliden-cyanoacetic acid 4-trifluoromethyl anilide), which is the active metabolite of the isoxazole derivative leflunomide [5-methyl-N-(4-trifluoromethyl-phenyl)-4-isoxazole carboximide]. An IC50 value of 1 microM was determined for A77 1726. Detailed kinetics experiments revealed uncompetitive inhibition with respect to dihydroorotate (Kiu = 0.94 microM) and non-competitive inhibition with respect to decylubiquinone (Kic = 1.09 microM, Kiu = 1.05 microM). These results suggest that the immunomodulating agent A77 1726 (currently in clinical phase III studies for the treatment of rheumatoid arthritis) is a very good inhibitor of human dihydroorotate dehydrogenase.

Amino Acid Sequence↗