Do mitochondria make nitric oxide? no?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO*) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO* production by the oxymyoglobin oxidation assay, or electrochemically using an NO* electrode. An apparent NO* production detected by the Griess assay was identified as an artifact. NO* generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO*. Our data show that NO* production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.     

Antioxidants Inhibit Angiogenesis In Vivo through Down-regulation of Nitric Oxide Synthase Expression and Activity

Although reactive oxygen species (ROS) participate in many cellular mechanisms, only few data exist concerning their involvement in physiological angiogenesis. The aim of the present work was to elucidate possible mechanisms through which ROS affect angiogenesis in vivo, using the model of the chicken embryo chorioallantoic membrane (CAM). Superoxide dismutase (SOD) and its membrane permeable mimetic tempol, dose dependently decreased angiogenesis and down-regulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. The NADPH oxidase inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and apocynin, but not allopurinol, also had a dose dependent inhibitory effect on angiogenesis and NO production in vivo. Catalase and the intracellular hydrogen peroxide (H²O²) scavenger sodium pyruvate decreased, while H²O² increased in a dose-dependent manner the number of CAM blood vessels, as well as the expression and activity of iNOS. Dexamethasone, which down-regulated NO production by iNOS and l-NAME, but not d-NAME, dose dependently decreased angiogenesis in vivo. These data suggest that antioxidants affect physiological angiogenesis in vivo, through regulation of NOS expression and activity.     

Twenty‐Four‐Hour Variation of L‐Arginine/Nitric Oxide/Cyclic Guanosine Monophosphate Pathway Demonstrated by the Mouse Visceral Pain Model

The L‐arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is known to be involved in central and peripheral nociceptive processes. This study evaluated the rhythmic pattern of the L‐arginine/NO/cGMP pathway using the mouse visceral pain model. Experiments were performed at six different times (1, 5, 9, 13, 17, and 21 h after light on) per day in male mice synchronized to a 12 h:12 h light‐dark cycle. Animals were injected s.c. with saline, 2 mg/kg L‐arginine (a NO precursor), 75 mg/kg L‐N^G‐nitroarginine methyl ester (L‐NAME, a NOS inhibitor), 40 mg/kg methylene blue (a soluble guanylyl cyclase and/or NOS inhibitor), or 0.1 mg/kg sodium nitroprusside (a nonenzymatic NO donor) 15 min before counting 2.5 mg/kg (i.p.) p‐benzoquinone (PBQ)‐induced abdominal constrictions for 15 min. Blood samples were collected after the test, and the nitrite concentration was determined in serum samples. L‐arginine or L‐NAME caused both antinociception and nociception, depending on the circadian time of their injection. The analgesic effect of methylene blue or sodium nitroprusside exhibited significant biological time‐dependent differences in PBQ‐induced abdominal constrictions. Serum nitrite levels also displayed a significant 24 h variation in mice injected with PBQ, L‐NAME, methylene blue, or sodium nitroprusside, but not saline or L‐arginine. These results suggest that components of L‐arginine/NO/cGMP pathway exhibit biological time‐dependent effects on visceral nociceptive process.     

Production of l-rhamnulose,a rare sugar,from l-rhamnose using commercial immobilized glucose isomerase

Abstract

A commercial immobilized d-glucose isomerase from Streptomyces murines (Sweetzyme) was used to produce l-rhamnulose from l-rhamnose in a packed-bed reactor. The optimal conditions for l-rhamnulose production from l-rhamnose were determined as pH 8.0, 60?°C, 300?g L^?1 l-rhamnose as a substrate, and 0.6?h^?1 dilution rate. The half-life of the immobilized enzyme at 60?°C was 809?h. Under the optimal conditions, the immobilized enzyme produced an average of 135?g L^?1 l-rhamnulose from 300?g L^?1 l-rhamnose after 16 days at pH 8.0, 60?°C, and 0.6?h^?1 dilution rate, with a productivity of 81?g/L/h and a conversion yield of 45% in a packed-bed reactor.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

Production of l-Threonine by Analog-resistant Mutants

Mutants resistant to α-amino-β-hydroxyvaleri0c acid (AHV) were derived from various bacteria which belong to Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium, or Bacillus by mutational treatment with N-methyl-N′-nitro-N-nitrosoguanidine(NTG), and screened for their ability to produce l-threonine. A number of l-threonine producers were obtained from each group of bacteria. Among them, the mutants derived from C. glutamicum KY9159(Met^?) were further mutagenized with NTG to derive thialysine(S-Lys)-resistant mutants. An AHV-resistant mutant, KY10484 was proved to be much more sensitive to the growth inhibition by thialysine than the parent strain, KY9159. From KY10484, a number of AHV- and thialysine-resistant mutants were derived. Approximately a half of these mutants were found to produce more l-threonine than KY10484. Among these mutants, KY10440 (Met^?, AHV^R, s-Lys^R) was used to investigate the cultural conditions for l-threonine production. The growth of KY10440 decreased largely with addition of l-homoserine, a threonine precursor. l-Asparagine, l-cystine, l-glutamine or l-arginine partially reversed the inhibitory effect of l-homoserine. Addition of these amino acids at low level led to increase l-threonine production. The amount of l-threonine accumulation reached to a level of 14mg/ml with a medium containing 10% glucose and to a level of 10 mg/ml with a medium containing 5% molasses (as glucose).

Another AHV- and thialysine-resistant mutant, KY10251 which was also derived from KY9159 was found to produce both 9 mg/ml of l-threonine and 5.5 mg/ml of l-lysine in a culture broth.     

Properties of Tyrosinase from Pseudomonas melanogenum

The properties of the tyrosinase from Pseudomonas melanogenum was investigated with the crude enzyme preparation. Optimum temperature and pH of the enzyme were 23°C and 6.8, respectively. l-Tyrosine, d-tyrosine, m-tyrosine, N-acetyl-l-tyrosine and l-DOPA were utilized as a substrate by the enzyme. The value for Km obtained were as follows: l-tyrosine 6.90 × 10^?4 m, d-tyrosine 1.43 ×10^?3 m and l-DOPA 9.90 × 10^?4 m. The enzyme was inhibited by chelating agents of Cu²⁺ l-cysteine, l-homocysteine, thiourea and diethyl-dithiocarbamate and the inhibition was completely reversed by the addition of excess Cu²⁺ From these results it is concluded that the enzyme is a copper-containing oxidase.     

Oxidative stress,nitric oxide,endothelial dysfunction and tinnitus

To assess whether pathogenic endothelial dysfunction is involved in acute idiopathic tinnitus we enrolled 44 patients and 25 healthy volunteers. In blood from the internal jugular vein and brachial vein we determined malonaldehyde, 4-hydroxynonenal, mieloperoxidase, glutathione peroxidase, nitric oxide, l-arginine and l-ornitine, thrombomodulin (TM) and von Willebrand factor (vWF) activity during tinnitus and asymptomatic period.

Higher plasma concentrations of oxidative markers and l-arginine, and lower nitric oxide and l-ornitine levels were observed in jugular blood of patients with tinnitus, there being a significant difference between brachial and jugular veins. TM and vWF activity were significantly higher in patients' jugular blood than in brachial blood.

Our results suggest oxidant, TM, vWF activity production are increased and nitric oxide production reduced in brain circulation reflux blood of patients with acute tinnitus. These conditions are able to cause a general cerebro-vascular endothelial dysfunction, which in turn induce a dysfunction of microcirculation in the inner ear.     

Phyllostine,a New Phytotoxic Compound Produced by Phyllosticta sp.

Ethionine-resistant mutants derived from Corynebacterium glutamicum KY 9276 (Thr^?) were found to accumulate l-methionine in culture media. One of the mutants, ER-107-4, which produced 250 μg/ml of l-methionine was subjected to further mutagenesis to obtain better l-methionine producers. l-Methionine production increased stepwise by successive endowing such markers as selenomethionine, 1,2,4-triazole, trifluoromethionine and methionine hydroxamate resistance. Thus, a mutant multi-resistant to ethionine, selenomethionine and methionine hydroxamate, ESLMR-724, produced 2 mg/ml of l-methionine in a medium containing 10% glucose.

Increase of l-methionine production was accompanied by increased levels and reduced repressibility of methionine-forming enzymes. The levels of methionine enzymes in ESLMR-724 increased to 2.5~4.2 fold of those in KY9276, In addition, homoserine-O-trans-acetylase and cystathionine γ-synthase which were strongly repressed by l-methionine in KY 9276 were stimulated by exogenous l-methionine in ESLMR-724. Implications of these results were discussed in relation to the productivity of l-methionine and the regulation of l-methionine biosynthesis.     

Structures of the Oligosaccharides from the Enzymic Hydrolyzate of Rice-straw Arabinoxylan by a Xylanase of Aspergillus niger

A trisaccharide consisting of two d-xylose units and one l-arabinose unit, and a tetrasaccharide consisting of three d-xylose units and one l-arabinose unit were isolated from the hydrolyzate of rice-straw arabinoxylan by the xylanase I produced by Asp. niger.

The structures of the trisaccharide and the tetrasaccharide were determined to be 3¹-α-l-arabinofuranosylxylobiose ([α]_d^? 80°) and 3¹-α-l-arabinofuranosylxylotriose ([α]_d^? 84°), respectively, by chemical and enzymic methods.

According to the structures of two arabinose-xylose mixed oligosaccharides, it was shown that the rice-straw arabinoxylan is composed of chain of 1,4-linked βd-xylopyranose residues and some of xylose residues have side-chain of 1,3-linked α-l-arabinofuranose.     

Elevated Body Fat in Rats by the Dietary Nitric Oxide Synthase Inhibitor,L-Nω Nitroarginine

The influence of the dietary nitric oxide (NO) synthase inhibitor, L-N^ω nitroarginine (L-NNA) on body fat was examined in rats. In experiment 1, all rats were fed with the same amount of diet with or without 0.02% L-NNA for 8 wk. L-NNA intake caused elevations in serum triglyceride and body fat, and reduction in serum nitrate (a metabolite of nitric oxide). The activity of hepatic carnitine palmitoyltransferase was reduced by L-NNA. In experiment 2, rats were fed for 8 wk with the same amount of diets with or without 0.02% L-NNA supplemented or not with 4% L-arginine. The elevation in body fat, and the reductions in serum nitrate and in the activity of hepatic carnitine palmitoyltransfererase by L-NNA were all suppressed by supplemental L-arginine. The results suggest that lower NO generation elevated not only serum triglyceride, but also body fat by reduced fatty acid oxidation.     

Amino Acid Metabolism in Microorganisms

3-Methylthiopropylamine (MTPA) formation from l-methionine in Streptomyces sp. K37 was studied in detail. The reaction was confirmed to be catalyzed by the decarboxylase of l-methionine. The properties of the enzyme were studied in detail using acetone dried cells or cell-free extract. The enzyme was specific for l-methionine. Pyridoxal phosphate stimulated the reaction and protected the enzyme against heat inactivation. The optimum pH for the reaction was 6.0~8.0 and the optimum temperature was about 40°C. Carbonyl reagents (10^?2~10^?3 m) inhibited the reaction completely, and silver nitrate and mercuric chloride (10^?3~10^?4 m) markedly inhibited the reaction. Km value for the reaction was 1.21 × 10^?5 m. l-Methionine assay using the decarboxylase was attempted and was found to be applicable to practical use.     

Inhibition of l-Alanine Adding Enzyme by Glycine

l-Alanine adding enzymes from Bacillus subtilis and Bacillus cereus which catalyzed l-alanine incorporation into UDPMurNAc were partially purified and the properties of the enzymes were examined. The enzyme from B. subtilis was markedly stimulated by reducing agents including 2-mercaptoethanol, dithiothreitol, glutathione and cysteine. Mn²⁺ and Mg²⁺ activated l-alanine adding activity and their optimal concentrations were 2 to 5 mm and 10 mm, respectively. The optimum pH was 9.5 and the Km for l-alanine was 1.8×10^?4m. l-Alanine adding reaction was strongly inhibited by p-chloromercuribenzoate and N-ethyl-maleimide. Among glycine, l- and d-amino acids and glycine derivatives, glycine was the most effective inhibitor of the l-alanine adding reaction. The enzyme from B. cereus was more resistant to glycine than that from B. subtilis. Glycine was incorporated into UDPMurNAc in place of l-alanine, and the Ki for glycine was 4.2×l0^?3m with the enzyme from B. subtilis. From these data, the growth inhibition of bacteria by glycine is discussed.     

Development of an Enzyme-Linked Immunosorbent Assay System for the Determination of Asymmetric Dimethylarginine Using a Specific Monoclonal Antibody

We produced a monoclonal antibody (mAb) against N ^G,N ^G-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM–100 nM ADMA, and ADMA levels in human plasma and urine were found to be 0.78 μM and 51.3 μmol/g of creatinine respectively.     

Purification and Properties of l-Arginase from Bacillus subtilis

l-Arginase (l-arginine amidinohydrolase, EC 3.5.3.1) was purified in a crystalline form from cells of Bacillus subtilis KY 3281 with an overall yield of 23.2%. The crystalline enzyme had a specific activity of 858 i.u./mg-protein and was ultracentrifugally homogeneous. It was estimated to have a molecular weight of 115,000±5000 by the method of Yphantis.

The enzyme highly specific for l-arginine showed the maximum activity at pH 10 with Mn²⁺ ion. The Km for l-arginine was 1.35 × 10^?2 m The activity was competitively inhibited by l-lysine, but not by l-ornithine and increased by the addition of Mn²⁺ or Co²⁺ ions. The stable pH and temperature ranges became wider in the presence of Mn²⁺ ion and l-threonine.     

Bacterial Racemization of α-Amino-ε-caprolactam

1. Several bacteria were isolated from soil which grew on both d- and l-aminolactam and whose cells had an activity to racemize them. They were identified as Achromobacter obae nov. sp., Achr. cycloclastes, Alcaligenes faecalis and Flavobacterium arborescens.

2. Racemization of d- and l-aminolactam was investigated using the lyophilized cells of Achr. obae nov. sp. The optimum pH value of the reaction was about 8.0. The racemizing activity was completely inhibited by 10^?4 m hydroxylamine, and the inhibition was removed by 10^?4 m pyridoxal phosphate. Five percent d- and l-aminolactam solutions were completely racemized with a concomitant slight formation of l-lysine.     

Mechanism of l-Threonine and l-Lysine Production by Analog-resistant Mutants of Corynebactemum glutamicum

Homoserine dehydrogenases and aspartokinases in l-threonine- or l-threonine and l-lysine-producing mutants derived from Corynebacterium glutamicum KY 9159 (Met^?) were studied with respect to the sensitivity to the inhibition by end products, l-threonine and l-lysine. The activities of homoserine dehydrogenases in the mutants which produced l-threonine or l-threonine and l-lysine were slightly less susceptible to the inhibition by l-threonine than the activity in the parent strain, KY 9159. The aspartokinases in the threonine-producing mutants, KY 10484 and KY 10230, which were resistant to α-amino-β-hydroxylvaleric acid (AHV, a threonine analog) and more sensitive to thialysine (a lysine analog) than the parent, were sensitive to the concerted feedback inhibition by l-lysine and l-threonine by about the same degree as KY 9159. The aspartokinase in an AHV- and thialysine-resistant mutant, KY 10440, which was derived from KY 10484 and produced about 14 mg/ml of l-threonine in a medium containing 10% glucose was less susceptible to the concerted feedback inhibition than KY 10484 or KY 9159, although the activity was still under the feedback control. In the parent strain, l-threonine activated aspartokinase activity in the absence of ammonium sulfate, an activator of the enzyme, but partially inhibited the activity in the presence of the salt. On the other hand, the enzyme of KY 10440 was activated by l-threonine either in the presence or in the absence of the salt. In another AHV- and thialysine-resistant mutant, KY 10251, which was derived from KY 10230 and produced both 9 mg/ml of l-threonine and 5/5 mg/ml of l-lysine, l-threonine and l-lysine simultaneously added hardly inhibited the activity of aspartokinase.

Implications of these results are discussed in relation to l-threonine or l-lysine production, AHV or thialysine resistance and regulation of l-threonine biosynthesis in these mutants.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

The present investigation is concerned with l-glutamic acid production in the presence of pyrrolidone carboxylic acid and glucose in Bacillus megaterium st. 6126. This strain does not grow on dl-pyrrolidone carboxylic acid (dl-PCA)¹⁾ as the sole source of carbon and nitrogen. The optimal concentration of yeast extract required for the maximal production of l-glutamic acid was 0.005% under the conditions used. As the yeast extract concentration was increased, growth increased proportionally; but the l-glutamic acid production did not exceed the control’s to which glucose and ammonium chloride had been added. l-Glutamic acid produced by both growing cultures and resting cells was derived from glucose and ammonium salt of dl-PCA. Isotope experiments suggested that the l-glutamic acid produced was partially derived from ammonium salt of dl-PCA in the growing culture which had been supplemented with d-glucose-U-¹⁴C or dl-PCA-1-¹⁴C and that ammonium salt of dl-PCA was consumed as the source of nitrogen and carbon for l-glutamic acid.     

Production of l-aspartic acid by biotransformation and recovery using reverse micelle and gas hydrate methods

l-Aspartic acid (l-Asp) was produced using Escherichia coli (ATCC 11303), and its recovery from the reaction mixture was studied using reverse micelle and gas hydrate methods. The effect of initial substrate concentration on l-Asp production was also investigated, and inhibition was shown to occur above 0.75 mol L^?1. The values of the kinetic constants were determined as r_max=2.33×10^?4 mol L^?1 min^?1, K_M=0.19 mol L^?1, and K_ss=3.98 mol L^?1. The reverse micelle phase used for extraction contained Aliquat-336, 1-decanol and isooctane, and a micro-injection technique was used for extraction of l-Asp. The reverse micelle system is a useful technique for obtaining small particle sizes, which can be used for the synthesis of nanoparticle biomolecules. Recovery of l-Asp from reverse micelles using CO₂ hydrates was carried out, giving a recovery of 55%. The formation of CO₂ hydrate from the reverse micelle solution breaks the micelle by reducing the amount of water in the micelle structure, thus precipitating the l-Asp.