Stoichiometric Conversion Research Articles

Physiological concentrations of melatonin inhibit nitric oxide synthase in rat cerebellum

In the present paper we show the inhibitory effect of melatonin on rat cerebellar nitric oxide synthase (NOS) activity. NO production was monitored by the stoichiometric conversion of L-arginine to L-citrulline. The inhibitory effect of melatonin was dose-dependent, with an IC 50 value of about 0.1 mM. However, a significant inhibition of enzyme activity (> 22%) was observed at 1 nM melatonin which is in the range of the physiological serum concentration of the hormone at night. The inhibitory effect of melatonin was observed exclusively in the presence of Ca ++. Results suggest a new and important role of the pineal hormone melatonin on central nervous system processes, i.e., by modulating NO production.

A telemetry-instrumentation system for monitoring multiple subcutaneously implanted glucose sensors.

An implantable potentiostat-radiotelemetry system for in vivo sensing of glucose is described. An enzyme electrode sensor measures the oxidation current of hydrogen peroxide formed by the stoichiometric conversion of glucose substrate and oxygen cofactor in an immobilized glucose oxidase layer. The sensor current is converted to a frequency and transmitted at programmable intervals (4, 32, 256 s) to a remote receiver. Low power CMOS circuitry is employed and device operation for up to 1.5 years is predicted using two series connected 250 mAh lithium cells. Crystal controlled RF frequencies uniquely identify each sensor allowing over 10 sensors within the same 10 m radius. A custom interface card allows a PC to program the receiver and handle the transmitted sensor data using software written in Microsoft C and QuickBasic. Software control allows on-the-fly sensor addition or subtraction to the sensor group being monitored. Over 10 sensors can be tracked long-term using the longest transmit interval, or four sensors can be tracked during short-term infusion studies when the transmit interval is reduced to 4 s. The design, construction, operation, and performance of the system hardware and software are described and evaluated.

Microbial Alkene Epoxidation—Merits and Limitations

The direct epoxidation of olefins using monooxygenases offers the realistic prospect of stoichiometric conversion of a pro-chiral substrate into an enantiomerically pure product. A number of microbial systems are available in which the mechanism of oxygen activation, and consequently the oxygen reactivity, differs. This paper discusses some of the merits and potential problems associated with these different systems.

Reductive dehalogenation of the trichloromethyl group of nitrapyrin by the ammonia-oxidizing bacterium Nitrosomonas europaea.

Suspensions of Nitrosomonas europaea catalyzed the reductive dehalogenation of the commercial nitrification inhibitor nitrapyrin (2-chloro-6-trichloromethylpyridine). The product of the reaction was identified as 2-chloro-6-dichloromethylpyridine by its mass fragmentation and nuclear magnetic resonance spectra. A small amount of 2-chloro-6-dichloromethylpyridine accumulated during the conversion of nitrapyrin to 6-chloropicolinic acid in an aerated solution in the presence of ammonia (T. Vannelli and A.B. Hooper, Appl. Environ. Microbiol. 58:2321-2325, 1992). Nearly stoichiometric conversion of nitrapyrin to 2-chloro-6-dichloromethylpyridine occurred at very low oxygen concentrations and in the presence of hydrazine as a source of electrons. Under these conditions the turnover rate was 0.37 nmol of nitrapyrin per min per mg of protein. Two specific inhibitors of ammonia oxidation, acetylene and allylthiourea, inhibited the rate of the dehalogenation reaction by 80 and 84%, respectively. In the presence of D2O, all 2-chloro-6-dichloromethylpyridine produced in the reaction was deuterated at the methyl position. In an oxygenated solution and in the presence of ammonia or hydrazine, cells did not catalyze the oxidation of exogenously added 2-chloro-6-dichloromethylpyridine to 6-chloropicolinic acid. Thus, 2-chloro-6-dichloromethylpyridine is apparently not an intermediate in the aerobic production of 6-chloropicolinic acid from nitrapyrin.

Tetrahydrobiopterin-dependent nitrite oxidation to nitrate in isolated rat hepatocytes

We have found that isolated rat hepatocytes and cell extracts catalyze the stoichiometric conversion of nitrite (either as a product of the oxidation of endogenously synthesized nitric oxide or added as sodium nitrite) to nitrate, which in extracts requires the presence of tetrahydrobiopterin and is inhibited by cyanide but apparently not by carbon monoxide. The reaction is sensitive to heat denaturation and does not involve oxidation of NO 2 − by a peroxidative or radical oxygen mechanism. These results indicate the presence of a hitherto undescribed mammalian mechanism of inorganic nitrogen oxide oxidation that may be a protective mechanism against one potentially damaging effect of endogenous 'NO production (NO 2 − formation), and also that assays of NO formation based on NO 2 − determination alone may be an inaccurate measurement of this activity.

Metabolism of tetrathionate inThiobacillus acidophilus

Abstract Cell-free extracts of Thiobacillus acidophilus catalysed the stoichiometric conversion of tetrathionate to thiosulphate, sulphur and two protons. The pH optimum of the enzyme activity was 3.0 and its temperature optimum 40°C. The enzyme was unstable at 30 and 40°C, at which its activity decreased to zero within 100 and 20 h, respectively. Enzyme activity was not affected by incubation for 1 week on ice or by freezing and thawing of the extract. The K m for tetrathionate was 0.3 mM. Enzyme activity was stimulated by ammonium sulphate up to a concentration of 1M. The results indicate that trithionate hydrolase cannot account for the observed conversion of tetrathionate.

Flow-injection analysis with electrochemical detection for determination of salicylic acid in pharmaceutical preparations

An enzyme-based flow-injection system with electrochemical detection suitable for the determination of salicylic acid in pharmaceuticals is described. The method makes use of the enzyme salicylate hydroxylase (EC 1.14.13.1), which is covalently bound to glass beads and put into the reactor of a flow system. The enzyme catalyses the stoichiometric conversion of salicylic acid to catechol, which can then be detected amperometrically at +0.45 V (vs. Ag/AgCl/KClsat.). The electrode response is linearly proportional to the concentration of salicylic acid between 5 and 150 μg ml−1. The detection limit is 0.4 μg ml−1. This method is simple, rapid and specific. The assayed samples yielded relative standard deviations between 0.5 and 2.0% and recoveries between 93 and 98%.

Reactivity of various UO 3 modifications in the fluorination to UF 4 by Freon-12

Six uranium trioxide modifications, α, β, γ, amorphous, ζ, and δ-UO 3, were fluorinated directly to UF 4 by reaction with CCl 2F 2 at 753 K. The first four oxides needed various periods of time to attain stoichiometric conversion to UF 4 (99%); these are 4, 4, 2 and 3 h, respectively. The apparent differences are attributed to the degree of disorder in the oxide structure and to their specific surface areas. The latter two oxides (γ and δ-UO 3) reacted passively towards fluorination, which was discussed in the light of their surface structure. These two oxides showed neither uranyl bonding character nor residual surface water (molecular or OH-groups). The former effect leads to the presence of an odd oxygen and enhances a covalent character and to a weakening effect on the U-O uranyl bond. The latter effect (surface water) furnishes acid-base centers that catalyze the fluorination process.

In vitro hydrolysis of methyl acetate, a limitation in application of head-space gaschromatography in biological monitoring of exposure

Stoichiometric conversion of methyl acetate to methanol in vitro was detected when methyl acetate was incubated with blood for 2 to 8 h. The velocity of the reaction was so fast that almost all of methyl acetate disappeared in 8 h. The methanol formation was further confirmed by means of gaschromatography-mass spectrometry. The capacity to hydrolyze methyl acetate was evenly distributed in cellular and noncellular fractions of blood, but not in urine. The significance of the observation is discussed in relation to biological monitoring of exposure to industrial ester solvents by means of head-space gaschromatography of blood samples.

Dependence of the metabolism of nitric oxide (NO) in healthy human whole blood on the oxygenation of its red cell haemoglobin.

Plasma or whole venous or arterialized blood from healthy human donors was incubated with NO (50-300 microM), and the resulting formation of methaemoglobin (MetHb), nitrosyl haemoglobin (HbNO), and plasma nitrite and nitrate were measured. In plasma, NO was converted to nitrite and nitrate in a ratio of 5:1. In arterial blood (O2 sat. 94-99%) NO was almost quantitatively converted to nitrate and MetHb. No nitrite was detected and HbNO formation was low. In venous blood (O2 sat. 36-85%) more HbNO and less nitrate was formed, in comparison to arterialized blood. We propose that NO liberated from endothelium of conductance and resistance vessels is taken up by red blood cells and inactivated by HbO2 via stoichiometric conversion to MetHb and nitrate.

Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway.

Aminoimidazole riobnucleotide carboxylase, the sixth step in the purine biosynthetic pathway, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR). The gene products of the purE and purK genes (PurE and PurK, respectively) thought to be responsible for this activity have been overexpressed and the proteins purified to homogeneity. PurE separates from PurK in the first ammonium sulfate fractionation during the purification. No evidence for association of the two gene products under a variety of conditions using a variety of methods could be obtained. To facilitate the assay for CAIR production, the purC gene product, 5-aminoimidazole-4-N-succinylcarboxamide ribonucleotide (SAICAR) synthetase has also been overexpressed and purified to homogeneity. The activities of PurE, PurK, and PurE.PurK have been investigated. PurE alone is capable of catalyzing the conversion of AIR to CAIR 1 million times faster than the nonenzymatic rate. The Km for HCO3- in the PurE-dependent reaction is 110 mM! PurK possesses an ATPase activity that is dependent on the presence of AIR. No bicarbonate dependence on this reaction could be demonstrated (less than 100 microM), and AIR is not carboxylated during the hydrolysis of ATP. Incubation of a 1:1 mixture of PurE and PurK at low concentrations of bicarbonate (less than 100 microM) revealed that CAIR is produced but requires the stoichiometric conversion of ATP to ADP and Pi. No dependence on the concentration of HCO3- could be demonstrated. A new energy requirement in the purine biosynthetic pathway has been established.

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake.

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of L-malate to L-lactate and CO2 concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C4-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.

Rapid degradation of neurotensin by stimulated rat mast cells

A RIA towards neurotensin (NT) using C-terminal- and N-terminal-specific antisera was used to study degradation of this tridecapeptide by isolated rat mast cells. Incubation of NT (10 μM) with peritoneal or pleural mast cells resulted in a rapid loss of NT immunoreactivity (iNT), as measured by C-terminal-directed antiserum, with little effect on N-terminal iNT. The rate of the reaction was faster with pleural cells ( T 1 2 , 30 s) than with peritoneal cells ( T 1 2 , 180 s) and was > 10-fold slower in the presence of metabolic poisons. The enzyme(s) involved is most likely released from the cells during secretion, as NT was degraded by media conditioned by compound 48 80 - stimulated mast cells 40–60 times faster than by media from unstimulated cells. This degradation by conditioned media was concentration dependent, pH dependent, and temperature sensitive. HPLC analyses indicated a near stoichiometric conversion of NT to NT(1–12) (66%) and NT(1–11) (34%) after incubation for 10–30 s with conditioned media. By 30 min only NT(1–11) and NT(1–10) were present. Phenanthroline (1 mM), an inhibitor of carboxypeptidase, prevented the loss of C-terminal iNT and the generation of NT(1–12) and NT(1–11). While NT(1–12) was effective in releasing histamine from mast cells in vitro and increasing vascular permeability in vivo, NT(1–11) was not. These results suggest that carboxypeptidase-like enzyme(s) could modulate the level and form of NT-related peptides in various states involving activation of mast cells.

Comparison of the catalytic properties of phospholipase A2 from pancreas and venom using a continuous fluorescence displacement assay

Phospholipases A2 from pig pancreas and the venoms from bee, Naja naja and Crotalus atrox have been studied by using a new continuous fluorescence displacement assay that utilizes normal phospholipid substrates [Wilton (1990) Biochem. J. 266, 435-439]. With limiting amounts of substrate, the assay demonstrated stoichiometric conversion into products with both pancreatic and venom enzymes, and thus would allow phospholipid determination at concentrations down to about 0.1 microM. The substrate specificity of the enzyme was determined for the four enzymes in terms of both phospholipid head group and fatty acid selectivity. None of the enzymes demonstrated a preference for arachidonic acid-containing phospholipid under the conditions of this assay. No lag was observed with any enzyme with either phosphatidylcholine or phosphatidylglycerol as substrate. With dipalmitoyl-phosphatidylcholine as substrate, the assay clearly highlighted the different membrane-penetrating properties of the pancreatic and Naja naja enzymes and demonstrated maximal activity for the pancreatic enzyme in the region of the phase-transition temperature of this substrate, at about 35 degrees C.

Application of a fluorometric method to measure glutamate release from single retinal photoreceptors

Release of the endogenous transmitter, glutamate, was measured from individual cone photoreceptors using a microfluorometric technique. The assay for glutamate was conducted within the lumen of a suction pipette, and was based on the fluorometric measure of the production of NADH from NAD +. This reaction was catalyzed by glutamate dehydrogenase contained in the pipette. Upon introduction of glutamate to the pipette, an increase in the NADH fluorescence was observed, representing the stoichiometric conversion of glutamate to NADH. The fluorescent signal was quantified, allowing an estimate of glutamate release from a single cone upon depolarization. The release observed was elicited upon depolarization of the cell with extrinsic current, and was detectable simultaneous with stimulation of the cell. Depolarization-induced release of endogenous glutamate was from the synaptic pedicle of the cell, and this release decreased with subsequent stimulations. The decrease in the release could be briefly reversed by an increase in the depolarization current used, or by allowing the cell to rest for several minutes.

Quantitative studies of gas–solid reactions by powder X-ray diffraction: stoichiometric and catalytic conversion of CO to CO2over YBa2Cu3O6 +x

Using the in situ cell and other analytical facilities described in the preceding paper, we have carried out an in situ study of both the catalytic and stoichiometric conversion of carbon monoxide to carbon dioxide over the triple-layer defective perovskite YBa2Cu3O6 +x. Pre-treatment with CO was used to arrive at a well defined value of x. For both the catalytic and stoichiometric reactions a biphasic mixture of the orthorhombic and tetragonal forms of the non-stoichiometric oxide is observed. A ‘biphasic’, Rietveld refinement of the X-ray diffraction patterns was successfully employed: and it proved possible to determine the precise oxygen content of each phase during the course of the gas–solid reaction at elevated temperatures (300–360 °C). The details of the kinetics of the CO to CO2 transformation and the solid-state compositions associated with the process shed some light on the mechanism of catalytic reaction.

Sorption and microbial degradation of naphthalene in soil-water suspensions under denitrification conditions

The microbial degradation of naphthalene under denitrification conditions in soil-water suspensions was dependent on solute partitioning between soil and water. Soil-associated naphthalene was in equilibrium with aqueous-phase solute, with the rate of naphthalene degradation being mixed order with respect to aqueous concentration. The rate of degradation was modeled by coupling Michaelis-Menten kinetics for aqueous-phase solute with an intraaggregate radial diffusion model for naphthalene sorption and desorption with soil. It was shown by modeling and confirmed by experiment, for the soil suspension particle sizes employed in these tests, that the naphthalene sorption-desorption process was reversible and rapid compared to the rate of microbial degradation. The maximum rate of microbial degradation was proportional to the soil-to-water ratio and independent of nitrate concentration for initial nitrate concentrations greater than several hundred micrograms per gram of soil. Nitrate reduction was described by utilizing the total mass removal of naphthalene and a stoichiometric conversion factor. A coupled solute desorption-degradation model is presented for microbial degradation of hydrophobic organic compounds that are desorbed from porous soil aggregates, assuming sorbed solute is inaccessible to microorganisms and that the rate of solute release from the solid is rapid compared to the rate of retarded intraaggregate diffusion.

ChemInform Abstract: Stoichiometric Catalytic Decomposition of Nitric Oxide Over Cu‐ZSM‐5 Catalysts.

AbstractIt is demonstrated that complete, stoichiometric conversion of NO to N2 and O2 can be achieved over a Cu‐ZSM‐5 catalyst at 773 K using optimized conditions.

Purification and characterization of heme oxygenase from chick liver

A major inducible form of heme oxygenase (EC 1.14.99.3) was purified from liver microsomes of chicks pretreated with cadmium chloride. The purification involved solubilization of microsomes with Emulgen 913 and sodium cholate, followed by DEAE-Sephacel, carboxymethyl-cellulose (CM-52) and hydroxyapatite chromatography, and FPLC through Superose 6 and 12 columns operating in series. The final product gave a single band on silver-stained SDS/polyacrylamide gels (Mr = 33,000). Optimal conditions for measurement of activity of solubilized heme oxygenase were studied. In a reconstituted system containing purified heme oxygenase, NADPH-cytochrome reductase, biliverdin reductase and NADPH, the Km for free heme was 3.8 +/- 0.5 microM; for heme in the presence of bovine serum albumin (5 mol heme/3 mol albumin) the Km was 5.0 +/- 0.8 microM; and the Km for NADPH was 6.1 +/- 0.4 microM (all values mean +/- SD, n = 3). Oxygen concentration as low as 15 microM, with saturating concentrations of heme and NADPH, did not affect the reaction rate, indicating that the supply of oxygen is not involved in the physiological regulation of activity of the enzyme. The pH optimum of the reaction was 7.4; at 37 degrees C, the apparent Vmax was 580 +/- 44 nmol biliverdin.(mg protein)-1.min-1 and the molecular activity was 19.2 min-1. Biliverdin IXa was the sole biliverdin isomer formed. In the presence of purified biliverdin reductase, biliverdin was converted quantitatively to bilirubin. Addition of catalase to the reconstituted system decreased the breakdown of heme to non-biliverdin products and led to nearly stoichiometric conversion of heme to biliverdin. Activity of the enzyme in the reconstituted system was inhibited by metalloporphyrins in the following order of decreasing potency: tin mesoporphyrin greater than tin protoporphyrin greater than zinc protoporphyrin greater than manganese protoporphyrin greater than cobalt protoporphyrin. Protoporphyrin (3.3 or 6.6 microM) (and several other porphyrins) and metallic ions (100 microM) alone had little if any inhibitory effect, except for Hg2+ which inhibited by 67% at 10 microM and totally at 15 microM. Following partial cleavage, fragments of the purified enzyme were sequenced. Comparison of sequences to those derived from cDNA sequences for the major inducible rat and human heme oxygenase showed 69% and 76% similarities, respectively. The histidine residue at position 132 of rat heme oxygenase-1 and the residues (Lys128-Arg136) flanking His132 were conserved in all three enzymes, as well as in the corresponding portion of a fourth less highly similar rat enzyme, heme oxygenase-2.(ABSTRACT TRUNCATED AT 400 WORDS)

4-aminobutyrate is not available to bacteroids of cowpea Rhizobium MNF2030 in snake bean nodules

Laboratory cultures of cowpea Rhizobium MNF2030 grew on 4-aminobutyrate (GABA) as sole source of carbon and nitrogen. GABA transport was active since it was inhibited by carbonyl cyanide mchlorophenyl hydrazone and 2,4-dinitrophenol and cells developed a 400-fold concentration gradient across the cell membrane. Arsenite treatment of GABA-grown cells revealed stoichiometric conversion of GABA to pyruvate, indicating that 2-oxoglutarate is not an intermediate in GABA catabolism. GABA catabolism by cells of strain MNF2030 grown on GABA appreared to involve GABA transaminase, succinic semialdehyde dehydrogenase and malic enzyme; the first two enzymes were specifically induced by growth on GABA. The deamination process and removal of NH3 in cells catabolizing GABA involved GABA: 2-oxoglutarate transaminase; glutamate: oxaloacetate aminotransferase; asparate: pyruvate aminotransferase and alanine dehydrogenase.