Limb and skin abnormalities in mice lacking IKKalpha.

The gene encoding inhibitor of kappa B (IkappaB) kinase alpha (IKKalpha; also called IKK1) was disrupted by gene targeting. IKKalpha-deficient mice died perinatally. In IKKalpha-deficient fetuses, limb outgrowth was severely impaired despite unaffected skeletal development. The epidermal cells in IKKalpha-deficient fetuses were highly proliferative with dysregulated epidermal differentiation. In the basal layer, degradation of IkappaB and nuclear localization of nuclear factor kappa B (NF-kappaB) were not observed. Thus, IKKalpha is essential for NF-kappaB activation in the limb and skin during embryogenesis. In contrast, there was no impairment of NF-kappaB activation induced by either interleukin-1 or tumor necrosis factor-alpha in IKKalpha-deficient embryonic fibroblasts and thymocytes, indicating that IKKalpha is not essential for cytokine-induced activation of NF-kappaB.

[Long-term results of arterial switch operation].

Surgical results and late outcome in 202 patients who had undergone arterial switch operation from 1984 to 1997 were investigated. Actuarial survival was 90.6% at 10 years and 90.0% at 20 years. Fifty-two patients (25.7%) underwent reoperation for pulmonary stenosis and 7 patients (3.5%) had aortic valve replacement. Freedom from re-intervention was 71.9% at 10 years and 60.4% at 20 years. Using xeno-pericardial patch for pulmonary reconstruction was strong predictor for postoperative pulmonary stenosis. Coronary ischemic event was rare but some patients showed electorocardiogram (ECG) change on exercise and hypoplastic left coronary artery. Cardiopulmonary function was almost normal in long term survivors.

Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study

Quinohemoprotein-cytochrome c complex alcohol dehydrogenase (ADH) of acetic acid bacteria consists of three subunits, of which subunit I contains pyrroloquinoline quinone (PQQ) and heme c, and subunit II contains three heme c components. The PQQ and heme c components are believed to be involved in the intramolecular electron transfer from ethanol to ubiquinone. To study the intramolecular electron transfer in ADH of Acetobacter methanolicus, the redox potentials of heme c components were determined with ADH complex and the isolated subunits I and II of A. methanolicus, as well as hybrid ADH consisting of the subunit I/III complex of Gluconobacter suboxydans ADH and subunit II of A. methanolicus ADH. The redox potentials of hemes c in ADH complex were -130, 49, 188, and 188 mV at pH 7.0 and 24, 187, 190, and 255 mV at pH 4.5. In hybrid ADH, one of these heme c components was largely changed in the redox potential. Reduced ADH was fully oxidized with potassium ferricyanide, while ubiquinone oxidized the enzyme partially. The results indicate that electrons extracted from ethanol at PQQ site are transferred to ubiquinone via heme c in subunit I and two of the three hemes c in subunit II. Copyright 1998 Elsevier Science B.V.

New quinoproteins in oxidative fermentation.

Several quinoproteins have been newly indicated in acetic acid bacteria, all of which can be applied to fermentative or enzymatic production of useful materials by means of oxidative fermentation. (1) D-Arabitol dehydrogenase from Gluconobacter suboxydans IFO 3257 was purified from the bacterial membrane and found to be a versatile enzyme for oxidation of various substrates to the corresponding oxidation products. It is worthy of notice that the enzyme catalyzes D-gluconate oxidation to 5-keto-D-gluconate, whereas 2-keto-D-gluconate is produced by a flavoprotein D-gluconate dehydrogenase. (2) Membrane-bound cyclic alcohol dehydrogenase was solubilized and purified for the first time from Gluconobacter frateurii CHM 9. When compared with the cytosolic NAD-dependent cyclic alcohol dehydrogenase crystallized from the same strain, the reaction rate in cyclic alcohol oxidation by the membrane enzyme was 100 times stronger than the cytosolic NAD-dependent enzyme. The NAD-dependent enzyme makes no contribution to cyclic alcohol oxidation but contributes to the reduction of cyclic ketones to cyclic alcohols. (3) Meso-erythritol dehydrogenase has been purified from the membrane fraction of G. frateurii CHM 43. The typical properties of quinoproteins were indicated in many respects with the enzyme. It was found that the enzyme, growing cells and also the resting cells of the organism are very effective in producing L-erythrulose. Dihydroxyacetone can be replaced by L-erythrulose for cosmetics for those who are sensitive to dihydroxyacetone. (4) Two different membrane-bound D-sorbitol dehydrogenases were indicated in acetic acid bacteria. One enzyme contributing to L-sorbose production has been identified to be a quinoprotein, while another FAD-containing D-sorbitol dehydrogenase catalyzes D-sorbitol oxidation to D-fructose. D-Fructose production by the oxidative fermentation would be possible by the latter enzyme and it is superior to the well-established D-glucose isomerase, because the oxidative fermentation catalyzes irreversible one-way oxidation of D-sorbitol to D-fructose without any reaction equilibrium, unlike D-glucose isomerase. (5) Quinate dehydrogenase was found in several Gluconobacter strains and other aerobic bacteria like Pseudomonas and Acinetobacter strains. It has become possible to produce dehydroquinate, dehydroshikimate, and shikimate by oxidative fermentation. Quinate dehydrogenase was readily solubilized from the membrane fraction by alkylglucoside in the presence of 0.1 M KCl. A simple purification by hydrophobic chromatography gave a highly purified quinate dehydrogenase that was monodispersed and showed sufficient purity. When quinate dehydrogenase purification was done with Acinetobacter calcoaceticus AC3, which is unable to synthesize PQQ, purified inactive apo-quinate dehydrogenase appeared to be a dimer and it was converted to the monomeric active holo-quinate dehydrogenase by the addition of PQQ.

The quinohemoprotein alcohol dehydrogenase of Gluconobacter suboxydans has ubiquinol oxidation activity at a site different from the ubiquinone reduction site.

Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. In addition to the reduction of ubiquinone, ADHs of Gluconobacter suboxydans and Acetobacter aceti were shown to have a novel function in the oxidation of ubiquinol. The oxidation activity of ubiquinol was detected as an ubiquinol:ferricyanide oxidoreductase activity, which can be monitored by selected wavelength pairs at 273 and 298 nm with a dual-wavelength spectrophotometer. The ubiquinol oxidation activity of G. suboxydans ADH was shown to be two times higher in 'inactive ADH', whose ubiquinone reductase activity is 10 times lower, than with normal 'active' ADH. No activity could be detected in the isolated subunit II or subunit I/III complex, but activity was detectable in the reconstituted ADH complex. Inactive and active ADHs exhibited a 2-3-fold difference in their affinity to ubiquinol despite having the same affinity to ubiquinone. Furthermore, the ubiquinol oxidation site in ADH could be distinguished from the ubiquinone reduction site by differences in their sensitivity to ubiquinone-related inhibitors and by their substrate specificity with several ubiquinone analogues. Thus, the results strongly suggest that the reactions occur at different sites. Furthermore, in situ reconstitution experiments showed that ADH is able to accept electrons from ubiquinol present in Escherichia coli membranes, suggesting the ubiquinol oxidation activity of ADH has a physiological function. Thus, ADH of acetic acid bacteria, which has ubiquinone reduction activity, was shown to have a novel ubiquinol oxidation activity, of which the physiological function in the respiratory chain of the organism is also discussed.

Two amine oxidases from Aspergillus niger AKU 3302 contain topa quinone as the cofactor: unusual cofactor link to the glutamyl residue occurs only at one of the enzymes.

Amine oxidases (EC 1.4.3.6) from Aspergillus niger, AO-I (2 x 75 kDa) and AO-II (80 kDa), were examined to determine the cofactor structure. Inactivated with p-nitrophenylhydrazine, they showed absorption and fluorescence spectra similar to those published for other copper amine oxidases and to topa hydantoin p-nitrophenylhydrazone. After digestion by thermolysin and pronase, cofactor peptides were purified by HPLC and sequenced. For thermolytic peptides, a typical topa consensus sequence, Asn-X-Glu-Tyr, was obtained for AO-II, although in case of AO-I it overlapped with Val-Val-Ile-Glu-Pro-Tyr-Gly. For pronase peptides of AO-I, only the latter sequence was obtained. NMR and mass spectroscopy confirmed the residue X as topa p-nitrophenylhydrazone in AO-II and revealed the presence of a residue Z attached to the Glu in the peptide Val-Val-Ile-Glu(Z)-Pro of AO-I. This residue was separated from the peptide by hydrolysis and identified as a product derived from topa quinone. The data, together with amino-acid sequence of AO-I, confer strong evidence for topa quinone as the cofactor, bound in the typical consensus sequence. Raman spectra of the p-nitrophenylhydrazone derivative of AO-I and its pronase peptide showed essentially the same peaks matching to a model compound for topa p-nitrophenylhydrazone. However, there may exist an unusual ester link between the topa-404 and Glu-145 in the native enzyme.

Gene organization and transcriptional regulation of the gntRKU operon involved in gluconate uptake and catabolism of Escherichia coli.

We cloned and characterized the gntRKU operon encoding part of the GntI system involved in gluconate uptake and catabolism by Escherichia coli. The operon was shown to encode its repressor, a thermoresistant gluconate kinase, and a low affinity gluconate permease. CAT fusion analysis revealed that the operon has a promoter for gntR and another for gntKU, and that the gntR gene is constitutively expressed, while that of gntKU is regulated positively by the cAMP-CRP complex and negatively by GntR. Read-through transcription from the gntR promoter into gntK was decreased in the presence of GntR, although GntR did not repress its own promoter. In addition, transcriptional attenuation was observed after the gntK gene, so gntU expression is reduced presumably to modulate the production of the low affinity gluconate permease according to the available concentration of gluconate.

Characterization of the gntT gene encoding a high-affinity gluconate permease in Escherichia coli.

We characterized the gntT gene encoding a high-affinity gluconate permease of Escherichia coli K-12. Primer extension and lacZ-operon fusion analyses revealed that gntT has one strong and two weak promoters, all of which are regulated positively by cAMP-CRP and negatively by GntR. The weak promoters became constitutive when separated from the upstream region including the strong promoter that overlaps a putative GntR-binding sequence. Gluconate-specific uptake activity was observed with cells harboring the gntT plasmid clone, which was enhanced by the presence of gntK encoding gluconate kinase.

Purification and characterization of the Escherichia coli thermoresistant glucokinase encoded by the gntK gene.

A thermoresistant gluconokinase encoded by the gntK gene of Escherichia coli K-12 was purified and characterized. The Km values of the purified enzyme for gluconate and ATP are 42 microM and 123 microM, respectively, and the activity was not altered by the presence of pyruvate. The enzyme was shown to function as a dimer with two identical subunits of 18.4 kDa. These characteristics appear to be distinct from those of the gluconokinase reported by E.I. Vivas, A. Liendo, K. Dawidowicz, and T. Istúriz (1994) J. Basic. Microbiol. 16, 117-122.

Reactivity with ubiquinone of quinoprotein D-glucose dehydrogenase from Gluconobacter suboxydans.

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent oxidoreductase linked to the respiratory chain of a wide variety of bacteria. There is a controversy as to whether the glucose dehydrogenase is linked to the respiratory chain via ubiquinone or cytochrome b. In this study, it was shown that the glucose dehydrogenase of Gluconobacter suboxydans has the ability to react directly with ubiquinone. The enzyme purified from the membranes of G. suboxydans was able to react with ubiquinone homologues such as ubiquinone-1, -2, or -6 in detergent solution. Furthermore, in order to demonstrate the reactivity of the enzyme with native ubiquinone, ubiquinone-10, in the native membranous environment, the dehydrogenase was reconstituted together with cytochrome o, the terminal oxidase of the respiratory chain, into a phospholipid bilayer containing ubiquinone-10. The proteoliposomes thus reconstituted exhibited a reasonable glucose oxidase activity, the electron transfer reaction of which was able to generate a membrane potential and a pH gradient. Thus, D-glucose dehydrogenase of G. suboxydans has been demonstrated to donate electrons directly to ubiquinone in the respiratory chain.

Occurrence of old yellow enzyme in Gluconobacter suboxydans, and the cyclic regeneration of NADP.

Old yellow enzyme system has been found in the cytosol fraction of Gluconobacter suboxydans. This is the first time that the enzyme has been found in organisms other than yeast cells. Old yellow enzyme [EC 1.6.99.1], D-glucose-6-phosphate dehydrogenase [EC 1.1.1.49], and catalase were isolated and crystallized separately from the organism. The old yellow enzyme from G. suboxydans showed catalytic and physicochemical properties almost identical with those of the enzyme from yeast cells. NADPH was specifically oxidized by the old yellow enzyme and the reduced enzyme was spontaneously reoxidized by atmospheric oxygen. The old yellow enzyme from G. suboxydans also contained FMN as a prosthetic group, and two mol of FMN were found per mol of enzyme (molecular weight, 88,000 as determined by gel filtration). In the oxidation of D-glucose-6-phosphate to 6-phospho-D-gluconate, cyclic regeneration of NADP occurred smoothly in the presence of D-glucose-6-phosphate dehydrogenase and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.

Membrane-bound D-gluconate dehydrogenase from Pseudomonas aeruginosa. Purification and structure of cytochrome-binding form.

A membrane-bound D-gluconate dehydrogenase [EC 1.1.99.3] was solubilized from membranes of Pseudomonas aeruginosa and purified to a homogeneous state with the aid of detergents. The solubilized enzyme was a monomer in the presence of at least 0.1% Triton X-100, having a molecular weight of 138,000 on polyacrylamide gel electrophoresis or 124,000--131,000 on sucrose density gradient centrifugation. In the absence of Triton X-100, the enzyme became dimeric, having a molecular weight of 240,000--260,000 on sucrose density gradient centrifugation. Removal of Triton X-100 caused a decrease in enzyme activity. Enzyme activity was stimulated by addition of phospholipid, particularly cardiolipin, in the presence of Triton X-100. The enzyme had a cytochrome c1, c-554(551), which might be a diheme cytochrome, and it also contained a covalently bound flavin but not ubiquinone. In the presence of sodium dodecyl sulfate, the enzyme was dissociated into three components with molecular weights of 66,000, 50,000, and 22,000. The components of 66,000 and 50,000 daltons corresponded to a flavoprotein and cytochrome c1, respectively, but that of 22,000 dalton remained unclear as to its function.

Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on the membranes.

The outer and inner cytoplasmic membranes of Pseudomonas aeruginosa were separated as small and large membranes, respectively, from the cell envelope of this organism treated with lysozyme in Tris-chloride buffer containing sucrose and MgCl2 by differential centrifugation. The small membrane fraction contained predominantly 2-keto-3-deoxyoctonate (KDO), and little cytochromes or oxidase activities. The small membrane was composed of only 9 polypeptides and showed homogeneous small vesicles electron-microscopically. On the other hand, the large membrane fraction had high cytochrome contents and oxidase activities, and little KDO. The large membrane was composed of a number of polypeptides and showed large fragments or vesicles electron-microscopically. These results indicate that the small and large membranes are the outer and inner cytoplasmic membranes of P. aeruginosa, respectively. The isolated outer membrane showed a symmetrical protein peak with a density of 1.23 on sucrose density gradient centrifugation and the isolated inner membrane showed an unusually high density, probably due to association with ribosomes and extrinsic or loosely bound proteins. EDTA lowered the density of both membranes and caused lethal damage to the outer membrane, causing disintegration with the release of lipopolysaccharide (LPS), proteins and phospholipid.

Membrane-bound cytochromes c of Pseudomonas aeruginosa grown aerobically. Purification and characterization of cytochromes c-551 and c-555.

Membrane-bound cytochromes c of Pseudomonas aeruginosa grown aerobically were investigated. By detecting polypeptides with heme-catalyzing peroxidase activity on a sodium dodecyl sulfate polyacrylamide gel, four major (Band I, 33,000 daltons; II, 25,000; III, 20,000; IV, 16,000) and one minor (V, 11,500) hemoproteins were found in the membrane fraction, while one hemoprotein (VI, 8,200) was detected in a small amount in the cytosol fraction. All these hemoproteins (bands I to VI) appeared to be cytochromes c, because all bands were detected even after being treated with HCl-acetone. Of the membrane-bound cytochromes c, cytochromes c-551 (band IV) and c-555 (band V) were solubilized with Triton X-100 and purified by repeated DEAE-cellulose column chromatography. Both purified cytochromes c-551 and c-555 were monomeric and their molecular weights were estimated to be 16,400 and 11,500, respectively. Their respective midpoint potentials were 0.31 and 0.34 V, and their respective isoelectric points in the reduced form were 3.8 and 5.2. The purified cytochromes c-551 and c-555 were found to be clearly different from "soluble" cytochrome c-551, and might function in the membrane-bound aerobic respiratory chain of P. aeruginosa.

Evidence for electron transfer via ubiquinone between quinoproteins D-glucose dehydrogenase and alcohol dehydrogenase of Gluconobacter suboxydans.

Gluconobacter suboxydans contains membrane-bound D-glucose and alcohol dehydrogenases (GDH and ADH) as the primary dehydrogenases in the respiratory chain. These enzymes are known to be quinoproteins having pyrroloquinoline quinone as the prosthetic group. GDH reduces an artificial electron acceptor, ferricyanide, in the membrane, but not after solubilization with Triton X-100, while ADH can react with the electron acceptor even after solubilization and further purification. In this study, it has been shown that the ferricyanide reductase activity of GDH is restored by adding the supernatant solubilized with Triton X-100 to the residue, and also by incorporation of purified ADH into the membranes of an ADH-deficient strain. G. suboxydans var. alpha. In addition, the ferricyanide reductase activity of GDH was reconstituted in proteoliposomes from GDH, ADH, and ubiquinone-10. Thus, the results indicated that the electron transfer from GDH to ferricyanide was mediated by ubiquinone and ADH. The data also suggest that GDH and ADH transfer electrons mutually via ubiquinone in the respiratory chain.

Methanol and ethanol oxidase respiratory chains of the methylotrophic acetic acid bacterium, Acetobacter methanolicus.

Acetobacter methanolicus is a unique acetic acid bacterium which has a methanol oxidase respiratory chain, as seen in methylotrophs, in addition to its ethanol oxidase respiratory chain. In this study, the relationship between methanol and ethanol oxidase respiratory chains was investigated. The organism is able to grow by oxidizing several carbon sources, including methanol, glycerol, and glucose. Cells grown on methanol exhibited a high methanol-oxidizing activity and contained large amounts of methanol dehydrogenase and soluble cytochromes c. Cells grown on glycerol showed higher oxygen uptake rate and dehydrogenase activity with ethanol but little methanol-oxidizing activity. Furthermore, two different terminal oxidases, cytochrome c and ubiquinol oxidases, have been shown to be involved in the respiratory chain; cytochrome c oxidase predominates in cells grown on methanol while ubiquinol oxidase predominates in cells grown on glycerol. Both terminal oxidases could be solubilized from the membranes and separated from each other. The cytochrome c oxidase and the ubiquinol oxidase have been shown to be a cytochrome co and a cytochrome bo, respectively. Methanol-oxidizing activity was diminished by several treatments that disrupt the integrity of the cells. The activity of the intact cells was inhibited with NaCl and/or EDTA, which disturbed the interaction between methanol dehydrogenase and cytochrome c. Ethanol-oxidizing activity in the membranes was inhibited with 2-heptyl-4-hydroxyquinoline N-oxide, which inhibited ubiquinol oxidase but not cytochrome c oxidase. Alcohol dehydrogenase has been purified from the membranes of glycerol-grown cells and shown to reduce ubiquinone-10 as well as a short side-chain homologue in detergent solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Function of ubiquinone in the electron transport system of Pseudomonas aeruginosa grown aerobically.

The location and function of ubiquinone in the electron transport system of Pseudomonas aeruginosa grown aerobically were studied. The reduction level of ubiquinone in the intact membrane was 36-43% in the aerobic steady state and about 65% in the anaerobic state with one substrate, but the level in the anaerobic state reached to 81% with a mixture of several substrates. Complete removal of ubiquinone performed by extracting the lyophilized membrane particles with n-pentane containing acetone resulted in complete loss of all oxidase activities for glucose, gluconate, malate, succinate, and NADH. In the ubiquinone-depleted particles, neither cytochrome component was reduced by adding any substrate. Reincorporation of coenzyme Q9 into the depleted particles restored each oxidase activity to 60 to 80% of the original and reduction of cytochromes with substrates. The reduction kinetics of cytochromes and effect of inhibitors showed that coenzyme Q9 was incorporated at the original site in the electron transport system. Exogenous coenzyme Q2 increased gluconate and malate oxidase activities and decreased glucose oxidase activity, when French-pressed membrane vesicles but not spheroplasts were used. Oxidizing activity for reduced coenzyme Q2 was also detected in the pressed vesicles but not in the spheroplasts. The present results showed that ubiquinone was indispensable and located prior to cytochromes in the electron transport system. Furthermore, the homogeneity and sidedness of ubiquinone in the cytoplasmic membrane of the organism are also discussed.

Membrane-bound respiratory chain of Pseudomonas aeruginosa grown aerobically. A KCN-insensitive alternate oxidase chain and its energetics.

There was found to be a KCN-insensitive, alternate oxidase chain branching from the ordinary oxidase chain in the respiratory chain of Pseudomonas aeruginosa grown aerobically. The alternate oxidase activity was highly resistant to KCN, and had a lower affinity for oxygen than ordinary cytochrome oxidase did. The branching point of the alternate oxidase chain from the ordinary oxidase chain was shown to be localized behind cytochrome b. The KCN-insensitive alternate oxidase chain was inhibited slightly with antimycin A and intensively with 2-thenoyltrifluoroacetone. The former inhibited the respiration behind cytochrome b and the latter before cytochrome b. N,N,N',N'-Tetramethyl-p-phenylenediamine oxidase-negative mutant (T105) was prepared from P. aeruginosa. The mutant clearly lacked a functional ordinary cytochrome oxidase, but had the KCN-insensitive alternate oxidase chain and could grow aerobically. The KCN-insensitive alternate oxidase chain had a H+/O ratio of 4, suggesting the existence of two energy-coupling sites in the chain. Under the conditions where both ordinary oxidase and alternate oxidase chains were functioning, the H+/O ratio of the parent strain was 5.6. From these data, we also discuss the energetics of the ordinary oxidase chain in the respiratory chain of aerobic P. aeruginosa.

NADH dehydrogenase of Corynebacterium glutamicum. Purification of an NADH dehydrogenase II homolog able to oxidize NADPH.

NADPH oxidase activity, in addition to NADH oxidase activity, has been shown to be present in the respiratory chain of Corynebacterium glutamicum. In this study, we tried to purify NADPH oxidase and NADH dehydrogenase activities from the membranes of C. glutamicum. Both the enzyme activities were simultaneously purified in the same fraction, and the purified enzyme was shown to be a single polypeptide of 55 kDa. The N-terminal sequence of the enzyme was consistent with the sequence deduced from the NADH dehydrogenase gene of C. glutamicum, which has been sequenced and shown to be a homolog of NADH dehydrogenase II. In addition to high NADH-ubiquinone-1 oxidoreductase activity at neutral pH, the purified enzyme showed relatively high NADPH oxidase and NADPH-ubiquinone-1 oxidoreductase activities at acidic pH. Thus, NADH dehydrogenase of C. glutamicum was shown to be rather unique in having a relatively high reactivity toward NADPH.

Pyrroloquinoline quinone: excretion by methylotrophs and growth stimulation for microorganisms.

A marked excretion of pyrroloquinoline quinone (PQQ) by methylotrophs into the culture medium was observed when incubation was prolonged to the late stationary phase. When the organisms were growing vigorously in the early exponential phase, accumulation of PQQ was repressed at a low level. Some evidence was obtained that the excretion of PQQ is related to turnover of quinoproteins of the organisms. The growth stimulation of microorganisms by PQQ was demonstrated using Acetobacter aceti. The presence of PQQ even at the pg/ml level in the culture medium stimulated the bacterial growth by reducing the lag time. The growth stimulating effect of PQQ was observed only by the reduction of the lag time but not by increase in either the subsequent growth rate or the total cell yield. The results indicated that PQQ must have an important role in the initiation of cell reproduction.