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Biomedical subjects

M Ritter

Publications and source records attributed to M Ritter.

At least 37 records · Page 2Linked to original sources
PubMed2001

Adipophilin is a sensitive marker for lipid loading in human blood monocytes.

Adipophilin, a marker of lipid accumulation initially described in adipocytes, was recently shown to be induced in macrophage foam cells. We found that even freshly isolated blood monocytes express adipophilin and that the amount of adipophilin protein is variable in monocytes from different healthy individuals. However, the physiological expression of adipophilin does not correlate with the levels of free fatty acids, cholesterylesters or free cholesterol. Enzymatically modified low-density lipoprotein (E-LDL) induces rapid foam cell formation in monocytes and upregulates adipophilin mRNA and protein within 2 h of incubation. This rapid induction of adipophilin is accompanied by a significant increase of free fatty acids in monocytes incubated with E-LDL. Adipophilin facilitates the uptake of free fatty acids, and here we demonstrate that free fatty acids increase is related to the early upregulation of adipophilin expression in blood monocytes. Fatty acids are ligands for peroxisome proliferator-activated receptor-gamma (PPARgamma), and the upregulation of adipophilin mRNA by PPARgamma agonists like 15d-PGJ(2) and ciglitazone indicates that PPARgamma may mediate the induction of adipophilin expression in human blood monocytes.

Adult↗
PubMed2001

Lipoprotein (a) up-regulates the expression of the plasminogen activator inhibitor 2 in human blood monocytes.

Elevated plasma lipoprotein (a) (Lp[a]) and cardiac events show a modest but significant association in various clinical studies. However, the influence of high Lp(a) on the gene expression in blood monocytes as a major cell involved in atherogenesis is poorly described. To identify genes influenced by elevated serum Lp(a), the gene expression was analyzed on a complementary DNA microarray comparing monocytes from a patient with isolated Lp(a) hyperlipidemia and coronary heart disease with monocytes from a healthy blood donor with low Lp(a). By using this approach, numerous genes were found differentially expressed in patient-versus-control monocytes. Verification of these candidates by Northern blot analysis or semiquantitative polymerase chain reaction in monocytes from additional patients with Lp(a) hyperlipidemia and healthy blood donors with elevated Lp(a) confirmed a significant induction of plasminogen activator inhibitor type 2 (PAI-2) messenger RNA (mRNA) in monocytes from male, but not from female, individuals with high Lp(a), indicating that this observation is gender specific. This led also to increased intracellular and secreted PAI-2 protein in monocytes from male probands with Lp(a) hyperlipidemia. Plasminogen activator inhibitor type 1 (PAI-1) mRNA was found suppressed only in the patients' monocytes and not in healthy probands with high Lp(a) levels. Purified Lp(a) induced PAI-2 mRNA and protein and reduced PAI-1 expression in monocytes isolated from various controls. The finding that PAI-2 is elevated in monocytes from male patients with isolated Lp(a) hyperlipidemia and male healthy probands with high Lp(a) and that purified Lp(a) up-regulates PAI-2 in control monocytes in vitro indicate a direct, but gender-specific, effect of Lp(a) for the induction of PAI-2 expression.

Adult↗
PubMed2001

Interaction of CD163 with the regulatory subunit of casein kinase II (CKII) and dependence of CD163 signaling on CKII and protein kinase C.

CD163 is a recently identified member of the scavenger receptor cysteine-rich superfamily, which is expressed on peripheral blood monocytes and most tissue macrophages and is thought to play an important role in the regulation of the inflammatory response of these cells. Cross-linking of CD163 on glucocorticoid-stimulated macrophages results in the secretion of several proinflammatory cytokines, but the precise mechanism of CD163 mediated signal transduction is not understood. The existence of several CD163 isoforms, which differ in the structure of their cytoplasmic domains and putative phosphorylation sites, suggests that these isoforms also differ in their signaling mechanism. Using the Yeast Two-Hybrid system and further in vitro and in vivo studies, we identified the regulatory beta-subunit of casein kinase II (CKII), which specifically binds to the cytoplasmic domain of CD163 and its isoforms. We also found, that in vitro the CD163 isoforms differ in their association with the CKII holoenzyme and in the phosphorylation by CKII. Furthermore, we demonstrated that the cytoplasmic domains of CD163 variants are phosphorylated by PKC-alpha in vitro. Inhibition studies using specific kinase inhibitors reveal that both CKII and PKC are involved in the CD163 signaling mechanism resulting in the secretion of proinflammatory cytokines.

2,3-Diphosphoglycerate↗
PubMed2001

[Possibilities and limits of outpatient antibiotic therapy of infective endocarditis].

BACKGROUND: Infective endocarditis has a high morbidity and lethality. Therefore antibiotic treatment has to be intravenous to achieve high blood levels and has to last several weeks without an interruption of treatment at the weekends. PATIENT SELECTION FOR OUTPATIENT THERAPY: It is therefore crucial to select the patient group that is suited for an outpatient, antibiotic therapy very carefully. In general only hemodynamically stable patients without complications in whom the responsible organism has been identified should be considered. TREATMENT: From a pharmacological point of view intravenous or intramuscular application for the complete duration of therapy is obligatory. Endocarditis caused by penicillin-susceptible streptococci, the biggest group of organisms responsible for endocarditis, can be treated with Ceftriaxon once daily for 4 weeks. For other organisms there are at present no data available which support the feasibility of an outpatient therapy. In particular antibiotic therapy with a complex regimen for those organisms is not practicable for outpatient usage. Possibly, in the near future computer controlled pumps might overcome this disadvantage. LIMITS: Every outpatient therapy should be initiated under inpatient conditions and only after an initial response to the antibiotic therapy continued in an outpatient setting. Today reliable outpatient therapy and follow-up 7 days a week under the given outpatient infrastructure is problematic and remains an exception. However, considering cost-effectiveness outpatient as compared to inpatient antibiotic therapy could be an interesting economically advantageous alternative.

Adult↗
PubMed2001

The sodium pump modulates the influence of I(Na) on [Ca2+]i transients in mouse ventricular myocytes.

To investigate whether activity of the sarcolemmal Na pump modulates the influence of sodium current on excitation-contraction (E-C) coupling, we measured [Ca(2+)](i) transients (fluo-3) in single voltage-clamped mouse ventricular myocytes ([Na+](pip) = 15 or 0 mM) when the Na pump was activated (4.4 mM K(+)(o)) and during abrupt inhibition of the pump by exposure to 0 K with a rapid solution-switcher device. After induction of steady state [Ca2+](i) transients by conditioning voltage pulses (0.25 Hz), inhibition of the Na pump for 1.5 s immediately before and continuing during a voltage pulse (200 ms, -80 to 0 mV) caused a significant increase (15 +/- 2%; n = 16; p < 0.01) in peak systolic [Ca2+](i) when [Na+](pip) was 15 mM. In the absence of sodium current (I(Na), which was blocked by 60 microM tetrodotoxin (TTX)), inhibition of the Na pump immediately before and during a voltage pulse did not result in an increase in peak systolic [Ca2+](i). Abrupt blockade of I(Na) during a single test pulse with TTX caused a slight decrease in peak [Ca2+](i), whether the pump was active (9%) or inhibited (10%). With the reverse-mode Na/Ca exchange inhibited by KB-R 7943, inhibition of the Na pump failed to increase the magnitude of the peak systolic [Ca2+](i) (4 +/- 1%; p = NS) when [Na+](pip) was 15 mM. When [Na+](pip) was 0 mM, the amplitude of the peak systolic [Ca2+](i) was not altered by abrupt inhibition of the Na pump immediately before and during a voltage pulse. These findings in adult mouse ventricular myocytes indicate the Na pump can modulate the influence of I(Na) on E-C coupling in a single beat and provide additional evidence for the existence of Na fuzzy space, where [Na+] can significantly modulate Ca2+ influx via reverse Na/Ca exchange.

Animals↗
PubMed2001

Functional importance and caffeine sensitivity of ryanodine receptors in primary lymphocytes.

Calcium signaling patterns are important for the specific regulation of activation and effector function in lymphocytes. Studies of [Ca2+]i regulation in lymphocytes, including the involvement of ryanodine receptors (RyR) and the importance of caffeine-sensitive pools, have been carried out mainly in lymphocyte cell lines and the presence and functional importance of these pools in primary lymphocytes has not been addressed. Here we show by confocal microscopy that caffeine caused a prompt but transitory increase of [Ca2+]i in primary lymphocytes, an effect that was inhibited by pre-treatment with ryanodine. Furthermore, the increase of [Ca2+]i in CD4+ and CD8+ MLR T lymphocytes stimulated by 5 microg/ml concanavalin A was significantly inhibited by pretreatment with caffeine. In functional studies, caffeine decreased cytotoxicity against myocyte target cells which is probably related to an altered calcium signaling in CD8+ MLR lymphocytes. Caffeine also terminated spontaneous Ca2+ oscillations and induced a rise in [Ca2+]i in CD4- and CD8- MLR lymphocytes probably of B cell origin. These results demonstrate that caffeine alters Ca2+ signaling in primary lymphocytes, and suggest that RyR, probably the skeletal muscle receptor (RyR-1) and brain receptor (RyR-3), are involved in mediating this effect. It is also possible that blocking of inositol-1,4,5-triphosphate (IP3) receptors is involved in the effects of caffeine on lymphocyte activation.

Animals↗
PubMed2001

Sequential monitoring of chimerism and detection of minimal residual disease after allogeneic blood stem cell transplantation (BSCT) using multiplex PCR amplification of short tandem repeat-markers.

Sequential analysis of chimerism after allogeneic blood stem cell transplantation (BSCT) has been shown to be predictive for graft failure and relapse. We have explored the impact of a novel approach for the quantitative determination of chimerism using a commercial PCR assay with multiplex amplification of nine STR-loci and fluorescence detection. The feasibility was studied in 121 patients transplanted from related or unrelated donors. Follow-up investigation was performed in 88 patients. Twenty-eight of these patients had received a transplantation after dose-reduced conditioning therapy. Results were compared to data obtained by FISH analysis in a subgroup of patients receiving grafts from sex-mismatched donors. The analysis was possible in all patients, the median number of informative alleles was 4 (range 1-8) compared to 7 (range 1-9) in the related and unrelated situation, respectively. A good correlation was seen in 84 samples from 14 patients analyzed in parallel with STR-PCR and FISH. Decreasing values of donor chimerism were detected prior to or concomitantly with the occurrence of graft failure and relapse of disease in all patients investigated prospectively. Using FACS-sorted material, eg peripheral blood CD34+ cells, the assay permitted the detection of residual recipient cells with high sensitivity (down to one CD34+ Kasumi cell in 40,000 normal WBC). Evaluation of the inter-laboratory reproducibility revealed that in 20 samples analyzed in three different centers, the median coefficient of variation was 2.1% (range 0.7-9.6%). Taken together, the results support the use of the test as a valuable tool in the follow-up of patients undergoing allogeneic BSCT. In cases lacking PCR-detectable disease-specific gene products, this assay may represent an alternative to recently established real-time PCR methods.

Adolescent↗
PubMed2001

Mutations in ras proto-oncogenes are associated with lower mdr1 gene expression in adult acute myeloid leukaemia.

Mutations in ras genes have been found to be the most frequent genetic aberrations in adult myeloid leukaemia (AML). Some reports have shown an improved outcome of ras-mutated AML. In order to understand the biology of ras mutation in AML, we studied a cohort of patients treated in a prospective multicentre trial for ras mutational status and resistance gene expression. Blast samples from 162 adult patients with de novo or secondary AML were examined for resistance gene expression (mdr1, mrp1 and lrp) and ras mutations using reverse transcription-polymerase chain reaction and protein nucleic acid-competitive polymerase chain reaction strategies respectively. Ras mutations were confirmed using DNA sequencing. Ras mutations leading to an exchange of amino acids were found in 40 (25%) patients. Thirty AML patients had N-ras mutations and nine patients had K-ras mutations. One patient showed both N-ras and K-ras mutations. Resistance gene expression was positive for mdr1 in 30%, for mrp1 in 43% and for lrp in 62% of patients. There was a strong inverse correlation between the presence of ras mutation and mdr1 expression (P = 0.005). However, no significant difference was seen between patients with or without ras mutations and mrp1 or lrp expression. Whereas mdr1 expression was associated with a lower complete remission rate (P < 0.04), ras mutations had no significant influence on remission status. Neither ras mutation nor mdr1 expression had a significant impact on overall or disease-free survival to date. For the first time, there is evidence that activated ras genes are associated with lower mdr1 expression in AML.

ATP-Binding Cassette Transporters↗
PubMed2001

ATP-binding cassette transporter A1 (ABCA1) affects total body sterol metabolism.

BACKGROUND AND AIMS: Members of the family of ABC transporters are involved in different processes of sterol metabolism, and ABCA1 was recently identified as a key regulator of high-density lipoprotein (HDL) metabolism. Our aim was to further analyze the role of ABCA1 in cholesterol metabolism. METHODS: ABCA1-deficient mice (ABCA1-/-) and wild-type mice were compared for different aspects of sterol metabolism. Intestinal cholesterol absorption was determined by a dual stable isotope technique, and analysis of fecal, plasma, and tissue sterols was performed by gas chromatography/mass spectrometry. Key regulators of sterol metabolism were investigated by Northern and Western blot analyses or enzyme activity assays. RESULTS: ABCA1-disrupted sv129/C57BL/6 hybrid mice showed a significant reduction in intestinal cholesterol absorption. The decrease in cholesterol absorption was followed by an enhanced fecal loss of neutral sterols, whereas fecal bile acid excretion was not affected. Total body cholesterol synthesis was significantly increased, with enhanced 3-hydroxy-3-methyglutaryl-coenzyme A (HMG-CoA) reductase observed in adrenals and spleen. In addition, ABCA1-/- mice showed markedly increased concentrations of cholesterol precursors in the plasma, lung, intestine, and feces. Reduced HMG-CoA reductase messenger RNA and enzyme activity in the liver suggest that enhanced cholesterol synthesis in ABCA1-/- mice occurs in peripheral tissues rather than the liver. CONCLUSIONS: The metabolism of cholesterol and cholesterol precursors is markedly affected by a lack of ABCA1 function.

ATP Binding Cassette Transporter 1↗
PubMed2001

Early growth response-1 gene: potential radiation response gene marker in prostate cancer.

This study was undertaken to determine whether the transcription factor EGR-1 expression: (1) in the primary tumor, correlates with radiation response in terms of complete local tumor control with no evidence of disease or recurrence and no evidence of metastasis; (2) in the postirradiated biopsies correlates with residual tumor; and (3) correlates with the expression of Egr-1 target genes such as TP53, pRB, and Bax. The authors analyzed: (1) 25 pretreated surgically resected paraffin-embedded primary adenocarcinomas of the prostate for the presence of EGR-1 expression and mutation, and correlated this with clinical endpoints such as serum prostate-specific antigen levels and current clinical status; (2) 27 postirradiated biopsies of prostate for the presence of EGR-1 expression, and correlated these findings to the residual tumor status; and (3) 12 prospective prostate tumor specimens for EGR-1 expression and its target genes. EGR-1 expression was determined by immunohistochemistry and mutations were screened in two regions of the Egr-1 gene (trinucleotide AGC repeats in transactivation domain [TD] and poly A tract in 3'UTR) by polymerase chain reaction-single strand conformational polymorphism analysis. Of 25 patients, 18 patients showed expression of EGR-1. EGR-1 overexpression correlated with treatment failure. No correlation with EGR-1 overexpression and its target genes was found, which may indirectly suggest that overexpressed EGR-1 may lack transactivation function. In summary, EGR-1 overexpression in the mutant form may provide an indication of clinical failure (local recurrence or metastasis).

Adenocarcinoma↗
PubMed2001

Gene variants of the bactericidal/permeability increasing protein and lipopolysaccharide binding protein in sepsis patients: gender-specific genetic predisposition to sepsis.

OBJECTIVES: To determine whether the genotype frequencies of the five bi-allelic polymorphisms in the bactericidal/permeability increasing protein (BPI) (Lys216 --> Glu; PstI polymorphism in intron 5; silent mutation G545 --> C) and the lipopolysaccharide binding protein (LBP) (Cys98 --> Gly; Pro436 --> Leu) are associated with the incidence and lethality of sepsis. DESIGN: Case control study of patients with sepsis. SETTING: Intensive care units within university hospitals. PATIENTS: A total of 204 patients diagnosed with sepsis and 250 healthy blood donors. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Short DNA fragments containing the polymorphic sites of the LBP and BPI locus were amplified by the polymerase chain reaction or mismatched polymerase chain reaction. The individual polymorphisms were determined with the appropriate restriction enzyme digestions and subsequent agarose gel electrophoresis. The presence of LBP genotypes with the less frequent Gly98 allele was found to be associated with sepsis (p < .02) in male patients, but not in females. Patients which were homozygote for either of the rare Gly98 (n = 6) and/or Leu436 (n = 5) LBP alleles, furthermore, exclusively were nonsurvivors of sepsis. The genotype frequencies in the BPI gene did not differ between patients and control individuals. CONCLUSIONS: Our findings suggest that common polymorphisms in the gene for LBP in combination with male gender are associated with an increased risk for the development of sepsis and, furthermore, may be linked to an unfavorable outcome. These data support the important immunomodulatory role of LBP in Gram-negative sepsis and suggest that genetic testing may be helpful for the identification of patients with an unfavorable response to Gram-negative infection.

Acute-Phase Proteins↗
PubMed2001

Inhibin, activin, and follistatin in human fetal pituitary and gonadal physiology.

BACKGROUND: Activin has been previously demonstrated to directly stimulate the synthesis of gonadotropin-releasing hormone (GnRH) receptors and to increase follicle-stimulating hormone (FSH) secretion in nonhuman pituitary cell cultures (PCCs). Currently, knowledge of the physiological role of these peptides in primates is still far from complete. Moreover, several results in macaque monkeys failed to support an unequivocal role for inhibin in FSH suppression. Whereas the bioactivity of inhibin and activin has been demonstrated in rat PCCs, no data exist on human pituitary response to these peptides either in vivo or in vitro. METHODS: We studied the secretion of FSH and luteinizing hormone (LH) by dispersed human fetal pituitary cells from midtrimester abortions in response to recombinant human (rh-) activin-A, inhibin-A, and other secretagogues. After mechanical and enzymatic dispersion, the human fetal pituitary cells were cultured on an extracellular matrixlike-material-coated 24-well plate. After 3 days' incubation in serum-containing medium, the PCCs were washed and preincubated for 90 min in serum-free medium and incubated with activin-A, inhibin-A, TGF-beta, follistatin, sex steroids, and GnRH, in quadruplicate. RESULTS: Activin-A was a potent secretagogue for FSH secretion. GnRH (20 ng/ml) was more potent than rh-activin-A for LH secretion. Nevertheless, a significant increase in LH secretion into the medium was brought about by rh-activin-A. Inhibin decreased FSH and LH secretion, but the LH response to inhibin was less prominent than that of FSH. GnRH opposed the inhibitory effect of inhibin on LH secretion. In dynamic, short-term, repetitive exposure of fetal pituitary fragments to rh-activin-A (superfusion), we could not receive a similar increase in LH and FSH as in static incubations, as opposed to a short GnRH exposure. In addition to their endocrine, paracrine, and autocrine effects, and in addition to their role as possible markers, the TGF-beta superfamily members may affect embryogenesis and possibly immunomodulation of the embryo and fetus. The role of activin and inhibin as intragonadal regulators is hypothesized. The pro-alphaC inhibin precursor may act as an FSH receptor antagonist. CONCLUSIONS: Human fetal PCCs express the previously reported physiologic responses to activin and inhibin generated in nonhuman experiments on gonadotropin secretion in vitro and may serve as a physiologic model for studying human gonadotrope responses to the TGF-beta family of peptides.

Activins↗
PubMed2001

Effects of deletion of muscle LIM protein on myocyte function.

Muscle LIM protein (MLP) may serve as a scaffold protein on the actin-based cytoskeleton, and mice deficient in this protein (MLPKO) have been recently reported to develop dilated cardiomyopathy. To determine the causes of depressed contractility in this model, we measured intracellular Ca2+ concentration ([Ca2+]i) transients (fluo 3), cell shortening, L-type Ca2+ channel current (I(Ca,L)), Na/Ca exchanger current (I(Na/Ca)), and sarcoplasmic reticulum (SR) Ca content in left ventricular MLPKO myocytes. I(Ca,L)-voltage relationships, I(Na/Ca) density, and membrane capacitance did not differ between wild-type (WT) and MLPKO myocytes. The peak systolic [Ca2+]i was significantly increased in MLPKO myocytes (603 +/- 54 vs. 349 +/- 18 nM in WT myocytes). The decline of [Ca2+]i transients was accelerated in MLPKO myocytes, and SR Ca2+ content was increased by 21%, indicating that SR Ca2+-ATPase function is normal or enhanced in MLPKO myocytes. Confocal imaging of actin filaments stained with tetramethylrhodamine isothiocyanate-labeled phalloidin showed disorganization of myofibrils and abnormal alignment of Z bands, and fractional shortening was significantly diminished in MLPKO myocytes compared with that in WT myocytes at comparable peak [Ca2+]i. Thus a reduced [Ca2+]-induced shortening may be involved in the pathogenesis of myocardial dysfunction in this genetic model of heart failure.

Actin Cytoskeleton↗
PubMed2001

Na(+)/H(+)exchangers: linking osmotic dysequilibrium to modified cell function.

The Na(+)/H(+) exchangers (NHEs) are among the major ion transporters involved in cell volume regulation. NHE activation leads to a cellular influx of Na(+) ions and extrusion of H(+) ions, which are readily replenished from intracellular buffers. This will result in a net import of Na(+). In many systems NHE operates in parallel to Cl(-)/ HCO3(-) exchange, resulting in cellular uptake of NaCl. The influx of osmotically obliged water will consequently lead to cell swelling. This makes NHEs suitable to serve as powerful mechanisms for increasing cell volume (CV). The low volume threshold for NHE activation enables the cells to respond to very minute reductions of the CV. By the coupling to the export of H(+) ions cell volume regulatory NHE activation may lead to changes in intracellular pH. On the other hand NHEs are activated by a broad variety of ligands and by intracellular acidosis, which, in turn, may consequently lead to cell swelling. In addition, NHEs are linked to other intracellular proteins and structures, like e.g. the cytoskeleton, which themelves are involved in the regulation of numerous cellular processes. Therefore NHEs link CV regulation to a diversity of cellular functions, both in physiological and pathophysiological conditions. Six isoforms of the Na(+)/H(+) exchanger, termed NHE1--6, have been cloned so far. NHE 1--5 are located in the plasma membrane, whereas NHE6 is sorted to the mitochondrial membrane. NHE1 and NHE6 are the ubiquitously expressed isoforms. The expression of the isoforms NHE2 to NHE5 is restricted to specific tissues and the pattern of their expression, as well as their subcellular localization indicate that they fulfill specialized functions. Cell shrinkage induced activation has been shown for NHE1,2 and 4. In contrast, NHE3 is inhibited by cell shrinkage. In many cells several isoforms are present and assigned to specific membrane domains where they may serve a functional crosstalk between the different ion transporters.

Amino Acid Sequence↗
PubMed2001

Determination of protein-protein interactions of ICIn by the yeast two-hybrid system.

ICln is a ubiquitously expressed eukaryotic protein. Expression of the protein in Xenopus laevis oocytes, the knocking-down of the protein in fibroblasts, or the reconstitution of the protein in lipid bilayer led to the assumption that this protein is an ionic channel or a significant part thereof. However, other possible roles for ICln in potential regulatory mechanisms have been postulated, as diverse as regulator of cell morphology by interacting with the Skb1 protein and/or interaction with core spliceosomal proteins. Here we show that ICln is able to interact with SnRNP core proteins SmD1, SmD2, SmD3, SmX5 and SmB/B'.

Amino Acid Sequence↗
PubMed2000

Essential role of Glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by Wolinella succinogenes fumarate reductase.

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalyzed by the related enzyme succinate:quinone reductase (succinate dehydrogenase). In the previously determined structure of QFR from Wolinella succinogenes, the site of fumarate reduction in the flavoprotein subunit A of the enzyme was identified, but the site of menaquinol oxidation was not. In the crystal structure, the acidic residue Glu-66 of the membrane spanning, diheme-containing subunit C lines a cavity that could be occupied by the substrate menaquinol. Here we describe that, after replacement of Glu-C66 with Gln by site-directed mutagenesis, the resulting mutant is unable to grow on fumarate and the purified enzyme lacks quinol oxidation activity. X-ray crystal structure analysis of the Glu-C66-->Gln variant enzyme at 3.1-A resolution rules out any major structural changes compared with the wild-type enzyme. The oxidation-reduction potentials of the heme groups are not significantly affected. We conclude that Glu-C66 is an essential constituent of the menaquinol oxidation site. Because Glu-C66 is oriented toward a cavity leading to the periplasm, the release of two protons on menaquinol oxidation is expected to occur to the periplasm, whereas the uptake of two protons on fumarate reduction occurs from the cytoplasm. Thus our results indicate that the reaction catalyzed by W. succinogenes QFR generates a transmembrane electrochemical potential.

Amino Acid Sequence↗