Aluminum Elicits Exocellular Phosphatidylethanolamine Production in Pseudomonas fluorescens

Pseudomonas fluorescens ATCC 13525 was found to grow in a minimal mineral medium supplemented with millimolar amounts of aluminum, a known environmental toxicant. During the stationary phase of growth, the trivalent metal was localized in a phosphatidylethanolamine (PE)-containing residue. The concentration of PE in pellets ranged from 1.7 to 13.9 mg ml of culture(sup-1) in media supplemented with 1 to 30 mM aluminum. Although the gelatinous residue was observed during the stationary phase of growth, ultracentrifugation and dialysis experiments revealed that PE was produced from earlier stages of incubation and was associated with aluminum. A sharp diminution in the levels of PE and aluminum in the spent fluid was concomitant with the formation of the insoluble deposit. The aluminum content of the soluble cellular fraction increased during growth and reached an optimum of 1.85 mM of test metal at 45 h in cultures with 15 mM aluminum. Further incubation, however, led to a marked decrease in the cellular aluminum content, and during the stationary phase of growth, only trace amounts of the trivalent metal were detected in this fraction. When 45-h cells were incubated in fresh citrate medium, most of the intracellular aluminum was secreted in the spent fluid and citrate was rapidly consumed. Aluminum efflux was also observed in cultures in which d-glucose was substituted for citrate. However, no efflux of this trivalent metal was evident in media devoid of either citrate or d-glucose. Scanning electron microscopic studies and X-ray energy-dispersive analyses of the dialyzed supernatant aided in the visualization of nodule-like aluminum- and phosphorus-rich bodies associated with thread-like carbon-, oxygen-, and phosphorus-containing structures. Transmission electron microscopic and electron energy loss spectroscopic analyses revealed the presence of aluminum within bacteria after 45 h of incubation. Cells harvested after aluminum insolubilization did not shown aluminum inclusions. This aluminum-tolerant microbe may have potential application in bioremediation processes.     

Transposon-induced pigment-deficient mutants of Porphyromonas gingivalis

Abstract Pseudomonas fluorescens appeared to grow in a mineral medium containing strontium (5 mM) complexed to citrate, the sole source of carbon, without any evident inhibition in cellular yield. Atomic absorption studies showed that the divalent metal was excluded from the cytoplasmic component and was initially found in the supernatant fluid in soluble form(s). However, as growth progressed, strontium was immobilized as a light green extracellular residue. X-ray fluorescence spectroscopy, X-ray diffraction spectrometry, Fourier transform infrared spectroscopy and acid treatment revealed that this insoluble moiety was crystalline strontium carbonate. Scanning electron microscopy aided in the identification of rounded bodies associated with these strontium carbonate crystals. A three-fold increment in the level of dissolved carbon dioxide observed in the spent fluid from the strontium supplemented medium would imply that this moiety may be playing a pivotal role in the deposition of the mineral.     

31P nuclear magnetic resonance study of the effect of azide on xylose fermentation by Candida tropicalis.

Maximal ethanol production by Candida tropicalis grown on xylose was obtained at an oxygen transfer rate of 5 to 7 mmol/liter per h. Addition of 0.2 mM azide increased the ethanol yield by a factor of 3 to 4, based on the cell mass produced, and decreased the formation of the by-product xylitol by 80%. In the presence of azide, ethanol was reassimilated before the carbon source was depleted. At all oxygenation levels studied, azide caused 25 to 60% of the carbon to be lost, most probably as carbon dioxide. Identical spectra were obtained with 31P nuclear magnetic resonance spectroscopy performed on extracts of C. tropicalis grown on xylose in the absence and presence of azide. Azide lowered the levels of sugar phosphates. Enzymatic analysis showed extremely low levels of fructose 1,6-diphosphate compared with the levels obtained in the absence of azide, while the level of malate, a citric acid cycle intermediate, was not influenced by azide. 31P nuclear magnetic resonance spectroscopy performed on xylose-grown whole cells of C. tropicalis showed that azide lowered the intracellular pH, inhibited the uptake of external Pi, and decreased the buildup of polyphosphate in relation to results with untreated cells. Similar results were obtained with the uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), except that CCCP treatment led to extremely high levels of internal Pi. The dual effect of azide as a respiratory inhibitor and as an uncoupler is discussed with respect to the metabolism and product formation in xylose-assimilating C. tropicalis.     

31P nuclear magnetic resonance study of the effect of azide on xylose fermentation by Candida tropicalis

Maximal ethanol production by Candida tropicalis grown on xylose was obtained at an oxygen transfer rate of 5 to 7 mmol/liter per h. Addition of 0.2 mM azide increased the ethanol yield by a factor of 3 to 4, based on the cell mass produced, and decreased the formation of the by-product xylitol by 80%. In the presence of azide, ethanol was reassimilated before the carbon source was depleted. At all oxygenation levels studied, azide caused 25 to 60% of the carbon to be lost, most probably as carbon dioxide. Identical spectra were obtained with 31P nuclear magnetic resonance spectroscopy performed on extracts of C. tropicalis grown on xylose in the absence and presence of azide. Azide lowered the levels of sugar phosphates. Enzymatic analysis showed extremely low levels of fructose 1,6-diphosphate compared with the levels obtained in the absence of azide, while the level of malate, a citric acid cycle intermediate, was not influenced by azide. 31P nuclear magnetic resonance spectroscopy performed on xylose-grown whole cells of C. tropicalis showed that azide lowered the intracellular pH, inhibited the uptake of external Pi, and decreased the buildup of polyphosphate in relation to results with untreated cells. Similar results were obtained with the uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), except that CCCP treatment led to extremely high levels of internal Pi. The dual effect of azide as a respiratory inhibitor and as an uncoupler is discussed with respect to the metabolism and product formation in xylose-assimilating C. tropicalis.     

Some significance of soil organic phosphorus mineralization in the phosphorus nutrition of cocoa in Ghana

Summary 1. Soil samples from a 2² NP:KMg factorial experiment, in which yield response of cocoa to NP was highly correlated to organic phosphorus content of the 0–2 layer, were studied.2. It was found that the organic phosphorus content ranged from 46.0 to 69.5 per cent of the total phosphorus and was correlated to the percentage total nitrogen, total phosphorus, organic carbon and pH.3. Incubation of the soils at 50 per cent of their water holding capacity and a temperature of 27°C for periods of 14, 28, 42, 56, and 70 days resulted in the average mineralization of organic phosphorus equivalent to 4,0. 13.6, 38.2, 50.0, and 54.0 pounds P per acre respectively.4. More organic phosphorus was mineralized at 50°C than at 27° or 40°C.5. The percentage of organic phosphorus mineralized was generally higher in the plots where NP had been applied.6. Laboratory application of nitrogen or phosphorus to the soils before incubation resulted in greater mineralization. The effect of nitrogen and phosphorus together was greater than in the presence of nitrogen alone but only in a few cases was it greater than in the presence of phosphorus alone. The effect of nitrogen and phosphorus was not additive.7. It is concluded that although the organic phosphorus increased in the NP-treated plots, increased mineralization occurred concurrently; and the effect of nitrogen and phosphorus application in increasing the mineralization of the soil organic phosphorus during laboratory incubation explained the high correlation found between cocoa yield response and organic phosphorus content of the top soil.     

X-ray photoelectron spectroscopy surface analysis of aluminum ion stress in barley roots

X-ray photoelectron spectroscopy (XPS) has been used to analyze root surface changes when Dayton barley (Hordeum vulgare) (Al tolerant) and Kearney barley (Al sensitive) seedlings were grown in nutrient solution in the presence and absence of 37.0 micromolar Al. The electron spectra from root surfaces contained strong lines in order of decreasing intensity from organic forms of carbon, oxygen, and nitrogen and weak lines due to inorganic elements in the form of anions and cations on the surface. The surface composition of root tips from Kearney was C, 65.6%; 0, 26.8%; N, 4.4% and tips from Dayton was C, 72.7%; O, 23.6%; N, 1.9%, grown in the absence of aluminum. Electron lines characteristic of nitrate, potassium, chloride, phosphate were also present in the spectra from those roots. Dayton roots grown in the presence of 37.0 micromolar aluminum contained 2.1% aluminum while Kearney contained 1.3% aluminum. The ratio of aluminum to phosphate was close to 1.0. Dayton roots usually contained twice as much aluminum phosphate in the surface region as Kearney. Dayton may be less susceptible to Al toxic effects by accumulation of aluminum phosphate on the root surface which then acts as a barrier to the transport of aluminum into the interior of the roots.     

Exocellular phosphatidylethanolamine production and multiple-metal tolerance in Pseudomonas fluorescens

Abstract Pseudomonas fluorescens appeared to circumvent the challenge imposed by millimolar amounts of metals (5 mM Al³⁺, 5 mM Fe³⁺, 2 mM Ca²⁺, 1 mM Ga³⁺ and 3 mM Zn²⁺) by the formation of phosphatidylethanolamine. This lipid moiety constituted an important organic component of an insoluble gelatinous residue in which most of the test metals were immobilized at stationary phase of growth. Ultracentrifugation and dialysis experiments showed that the metals were associated with phosphatidylethanolamine from early stages of growth. Transmission electron microscopy revealed metal rich bodies in the cytoplasm prior to their secretion in the spent fluid. These results demonstrate a role of phosphatidylethanolamine in multiple-metal homeostasis.     

Dinitrophenol influences the rate and yield of Acetobacter methanolicus during the growth on glucose

Acetobacter methanolicuswas grown on glucose in the presence of dinitrophenol (DNP) under carbon/energy-limited conditions. DNP affected both the growth yield and the growth rate (D_sh) at which the energy generation was shifted from a complete to an incomplete substrate oxidation by using the PQQ-linked glucose dehydrogenase. The more the growth yield was decreased, the higher both the DNP concentration and the growth rate became. At about 0.53 mM DNP, growth was completely stopped. D_sh decreased from 0.21h^?1in the absence of DNP to 0.175 h^?1and 0.075 h^?1in the presence of 0.2 mM and 0.4 mM DNP, respectively. The experimental data are discussed in terms of the limitations in the generation of energy and some stress situations which are exerted by the presence of the uncoupler.     

trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid is superior to ethylenediamine-N,N,N',N'-tetraacetic acid for sequestering Mg2+ in 31P NMR experiments involving ATP spectra at neutral and acidic pH

The use of ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) to sequester Mg2+ from samples containing ATP at acidic or neutral pH prior to 31P NMR spectroscopic analysis leads to significant broadening of the gamma- and beta-phosphorus resonances of ATP as compared to ATP alone. It was found that the use of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) reduces the broadening of the ATP resonances. At pH 7.0, 30 mM EDTA in the presence of 5 mM ATP and 7 mM Mg2+ leads to a threefold increase in the peak width of the gamma phosphorus of ATP as compared to 5 mM ATP alone. When 30 mM CDTA is used in the place of EDTA, the peak width decreased to about 80% of the peak width of ATP alone. When the experiment is repeated at pH 8.5, both EDTA and CDTA lead to narrow peak widths with no significant difference between the two spectra. At pH 6.0, the use of EDTA leads to a spectrum that is very noisy, with a 10-fold increase in the peak width as compared to ATP in the absence of Mg2+ at this pH, whereas the increase with CDTA is only 50%. These results do not reflect the difference in chelating strength between EDTA and CDTA: The free Mg2+ concentration in the presence of each chelator, as calculated by the computer program given in the Appendix, was nearly equal at each pH. The results, however, reflect a difference in the lability of the metal-ligand bond between EDTA and CDTA.     

Inhibition of Na+-H+ exchange by N,N'-dicyclohexylcarbodiimide in isolated rat renal brush border membrane vesicles

The inactivation of rat renal brush border membrane Na+-H+ exchange by the covalent carboxylate reagent N,N'-dicyclohexylcarbodiimide (DCCD) was studied by measuring 1 mM Na+ influx in the presence of a pH gradient (pHi = 5.5; pHo = 7.5) and H+ influx in the presence of a Na+ or Li+ gradient ([Na+]i = 150 mM; [Na+]o = 1.5 mM). In the presence of DCCD, the rate of Na+ uptake decreased exponentially with time and transport inhibition was irreversible. At all DCCD concentrations the loss of activity was described by a single exponential, consistent with one critical DCCD-reactive residue within the Na+-H+ exchanger. Among several carbodiimides the most hydrophobic carbodiimide, DCCD, was also the most effective inhibitor of Na+-H+ exchange. With 40 nmol of DCCD/mg of protein, at 20 degrees C for 30 min, 75% of the amiloride-sensitive 1 mM Na+ uptake was inhibited. Neither the equilibrium Na+ content nor the amiloride-insensitive Na+ uptake was significantly altered by the treatment. The Na+-dependent H+ flux, measured by the change in acridine orange absorbance, was also decreased 80% by the same DCCD treatment. If 150 mM NaCl, 150 mM LiCl, or 1 mM amiloride was present during incubation of the brush border membranes with 40 nmol of DCCD/mg of protein, then Li+-dependent H+ flux was protected 50, 100, or 100%, respectively, compared to membranes treated with DCCD in the absence of Na+-H+ exchanger substrates. The combination of DCCD and an exogenous nucleophile, e.g. ethylenediamine and glycine methyl ester, increased Na+-dependent H+ flux in the presence of 80 nmol of DCCD/mg of protein, compared to the transport after DCCD treatment alone. These findings suggest that the Na+-H+ exchanger contains a single carboxylate residue in a hydrophobic region of the protein, and the carboxylate and/or a nearby endogenous nucleophilic group is critical for exchange activity.     

Influence of salt on the structure of DMPG studied by SAXS and optical microscopy

Aqueous dispersions of 50 mM dimyristoylphosphatidylglycerol (DMPG) in the presence of increasing salt concentrations (2-500 mM NaCl) were studied by small angle X-ray scattering (SAXS) and optical microscopy between 15 and 35 degrees C. SAXS data show the presence of a broad peak around q approximately 0.12 A(-1) at all temperatures and conditions, arising from the electron density contrasts within the bilayer. Up to 100 mM NaCl, this broad peak is the main feature observed in the gel and fluid phases. At higher ionic strength (250-500 mM NaCl), an incipient lamellar repeat distance around d=90-100 A is detected superimposed to the bilayer form factor. The data with high salt were fit and showed that the emergent Bragg peak is due to loose multilamellar structures, with the local order vanishing after approximately 4d. Optical microscopy revealed that up to 20 mM NaCl, DMPG is arranged in submicroscopic vesicles. Giant (loose) multilamellar vesicles (MLVs) start to appear with 50 mM NaCl, although most lipids are arranged in small vesicles. As the ionic strength increases, more and denser MLVs are seen, up to 500 mM NaCl, when MLVs are the prevailing structure. The DLVO theory could account for the experimentally found interbilayer distances.     

Inducible aluminum resistance of Acidiphilium cryptum and aluminum tolerance of other acidophilic bacteria

Aluminum ions are highly soluble in acidic environments. Toxicity of aluminum ions for heterotrophic, facultatively and obligately chemolithoautotrophic acidophilic bacteria was examined. Acidiphilium cryptum grew in glucose-mineral medium, pH 3, containing 300 mM aluminum sulfate [Al(2)(SO(4))(3)] after a lag phase of about 120 h with a doubling time of 7.6 h, as compared to 5.2 h of growth without aluminum. Precultivation with 1 mM Al(2)(SO(4))(3) and transfer to a medium with 300 mM Al(2)(SO(4))(3) reduced the lag phase from 120 to 60 h, and immediate growth was observed when A. cryptum was precultivated with 50 mM Al(2)(SO(4))(3), suggesting an aluminum-induced resistance. Aluminum resistance was not induced by Fe(3+) ions and divalent cations. Upon exposure of A. cryptum to 300 mM Al(2)(SO(4))(3), the protein profile changed significantly as determined by SDS-PAGE. When other acidophiles were cultivated with 50-200 mM aluminum sulfate, no lag phase was observed while the growth rates and the cellular yields were significantly reduced. This growth response was observed with Acidobacterium capsulatum, Acidiphilium acidophilum, Acidithiobacillus ferrooxidans, and Acidithiobacillus thiooxidans. Precultivation of these strains with aluminum ions did not alter the growth response caused by aluminum. The content of A. cryptum cultivated with 300 mM Al(2)(SO(4))(3)was 0.44 microg Al/mg cell dry weight, while that of the other strains cultivated with 50 mM Al(2)(SO(4))(3) ranged from 0.30 to 3.47 microg Al/mg cell dry weight.     

Overexpression, purification and characterization of the catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus gracilirostris

The luciferase secreted by the deep-sea shrimp Oplophorus consists of 19 and 35kDa proteins. The 19-kDa protein (19kOLase), the catalytic component of luminescence reaction, was expressed in Escherichia coli using the cold-shock inducted expression system. 19kOLase, expressed as inclusion bodies, was solubilized with 6M urea and purified by urea-nickel chelate affinity chromatography. The yield of 19kOLase was 16 mg from 400 ml of cultured cells. 19kOLase in 6M urea could be refolded rapidly by dilution with 50mM Tris-HCl (pH 7.8)-10mM EDTA, and the refolded protein showed luminescence activity. The luminescence properties of refolded 19kOLase were characterized, in comparison with native Oplophorus luciferase. Luminescence intensity with bisdeoxycoelenterazine as a substrate was stimulated in the presence of organic solvents. The 19kOLase is a thermolabile protein and is 98 % inhibited by 1muM Cu2+. The cysteine residue of 19kOLase is not essential for catalysis of the luminescence reaction.     

Regulation of autotrophic and heterotrophic metabolism in Pseudomonas oxalaticus OX1: Growth on mixtures of acetate and formate in continuous culture

Growth of Pseudomonas oxalaticus in carbon- and energy-limited continuous cultures with mixtures of acetate and formate resulted in the simultaneous utilization of both substrates at all dilution rates tested. During growth on these mixtures, acetate repressed the synthesis of ribulosebisphosphate carboxylase. The degree of this repression was dependent on the dilution rate and on the ratio of acetate and formate in the medium reservoir. At fixed acetate and formate concentrations in the inflowing medium of 30 and 100 mM, respectively, and dilution rates above 0.10h^-1, the severe repression of autotrophic enzymes resulted in a marked increase in bacterial dry weight compared to the growth yield of the organisms on the two substrates separately. Also, at these dilution rates a significant increase in isocitrate lyase activity was observed in the cells as compared to growth on acetate alone. This indicated that under these conditions more acetate was assimilated and less dissimilated since acetate was partly replaced by formate as the energy source. When formate was added to the reservoir of an acetate-limited culture (S_R=30 mM), derepression of RuBPCase synthesis was observed at formate concentrations of 50 mM and above. Below this concentration formate only served as an energy source for acetate assimilation; when its concentration was increased above 50 mM a progressively increasing contribution of carbon dioxide fixation to the total carbon assimilation was observed as the activity of RuBPCase in the cells increased. It is concluded that in Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic carbon dioxide fixation via the Calvin cycle is regulated by a repression/derepression mechanism.Abbreviations RuBPCase ribulosebisphosphate carboxylase - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - FDH formate dehydrogenase - S_R concentration of growth-limiting substrate in reservoir     

Mycological Applications of X-Ray Microanalysis

In Aspergillus fumigatus, X-ray microanalysis applied in wavelength dispersive spectroscopy enabled us to detect the presence of calcium, potassium, sulfur, phosphorus, and magnesium; and in A. niger we detected the presence of calcium, potassium, chloride, sulfur, phosphorus, sodium, and magnesium. These various elements were identified by their K alpha lines.     

Implications of soil organic carbon and the biogeochemistry of iron and aluminum on soil phosphorus distribution in flooded forests of the lower Orinoco River,Venezuela

Relationships among soil phosphorus distribution, soil organic carbon and biogeochemistry of iron and aluminum were studied along a flooded forest gradient of the Mapire river, Venezuela. Soil samples were collected during the dry season in three zones subjected to different flooding intensity: MAX inundated 8 months per year, MED inundated 5 months per year, and MIN inundated 2 months per year. Total labile phosphorus (resin + bicarbonate extractable fractions) was significantly higher in MIN than in MAX. The longer non-flooding period in MIN probably allowed a higher accumulation of microbial biomass in soils of this zone and consequently a greater release of the bicarbonate organic fraction. The moderately labile phosphorus fraction associated with the chemisorbed phosphorus on amorphous and some crystalline aluminum and iron was significantly lower in MAX than in MIN following the same tendency observed for crystalline iron oxides. This result allowed us to hypothesize that the combined effect of a long flood period and a high soil organic carbon content in the MAX, could be appropriate conditions for microbial reduction of stable forms of iron. The ratio of soil organic carbon to total organic phosphorus decreased from MAX to MIN, indicating higher mineralization of organic phosphorus in MIN. Our results suggests two distinct flood-dependent mechanisms operating for phosphorus release along the gradient. In MAX mineralization process appears to be limited, while microbial mineral dissolution appears to be an important source of phosphorus. In MIN supply of phosphorus is associated with the stability of soil organic matter.     

2-[(4-Bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate, a new nucleotide analogue that acts as an affinity label of pyruvate kinase

A new reactive adenine nucleotide has been synthesized: 2-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 5'-monophosphate (2-BDB-TAMP). Adenosine 5'-monophosphate 1-oxide was synthesized by reaction of AMP with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 5'-monophosphate (TAMP). The final product was generated by reaction of TAMP with 1,4-dibromobutanedione. The structure of 2-BDB-TAMP was determined by UV, 1H NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Rabbit muscle pyruvate kinase is inactivated by 2-BDB-TAMP at pH 7.0 and 25 degrees C. The inactivation rate exhibits a nonlinear dependence on the reagent concentration with KI = 0.57 mM. Protection against inactivation is provided by ADP and ATP, in the presence of Mn2+, as well as by phosphoenolpyruvate, in the presence of K+; in addition, partial protection is provided by AMP plus Mn2+. Incubation of pyruvate kinase with 0.075 mM 2-BDB-TAMP for 70 min in the absence of protective ligands leads to incorporation of 1.55 mol of reagent/mol of enzyme subunit when the enzyme is 53% inactive. In the presence of ADP and Mn2+, only 0.96 mol of reagent/mol of subunit is incorporated at 70 min, while the enzyme retains 100% activity. Similar results were obtained in the presence of ATP plus Mn2+. Assuming that the groups modified in the absence of ligands include those modified in the presence of the nucleotides, the 53% inactivation can be attributed to the modification of 0.59 (1.55-0.96) group per enzyme subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear Magnetic Resonance Studies on the Effects of Decreased External Sodium on Guinea Pig Cerebral Cortex Slices and the Permeabilities of Various Sodium Substitutes

Decreasing the external sodium concentration ([Na+]e) to 10 mM in the presence of 280 mM sucrose had no significant effect on phosphocreatine (PCr) or on intracellular pH (pHi) as assessed using 31P nuclear magnetic resonance spectroscopy. Zero [Na+]e in the presence of 300 mM sucrose caused a fall in PCr levels to 50% of control values, and the pHi fell to 6.85 from a control value of 7.30. 1H nuclear magnetic resonance spectroscopy confirmed that the sucrose had not entered the tissue. The decreases in PCr content and in pHi, known to occur on depolarization using 40 mM external potassium concentration ([K+]e), were further decreased in the presence of 10 mM [Na+]e), to 51.4 +/- 4.0 and 6.80 +/- 0.10% of control values, respectively. The free intracellular magnesium concentration was significantly increased from a control value of 0.37 +/- 0.10 mM to 0.66 +/- 0.13 mM (p less than 0.001), when [Na+]e was decreased to 10 mM, but was not further affected by high [K+]e or zero Na+. Membrane permeabilities of the sodium substitutes N-methyl-D-glucamine (NMG), tris(hydroxymethyl)aminomethane (Tris), tetramethylammonium (TMA), and choline were assessed using 1H nuclear magnetic resonance spectroscopy. In the presence of 10 mM [Na+]e, NMG, TMA, and choline (all at 140 mM) were taken up and remained within the tissue for at least 2 h, but no uptake of Tris (140 mM) or sucrose (above) could be detected. Tissue lactate levels (from the lactate/N-acetyl aspartate ratio) increased in the presence of the substitutes that were taken up, although no change in pH was detected.     

Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

(4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes.     

Determination of protein orientation on surfaces with X-ray photoelectron spectroscopy

The use of X-ray photoelectron spectroscopy (XPS) for determination of the preferred orientation of proteins on metal surfaces is demonstrated. A myoglobin derivative in which a pentaamineruthenium (III) group is attached to a specific histidine (residue 81) is used for this purpose, with the Ru and Fe acting as a double marker. The relative intensities of the XPS photoelectron spectra for these two markers are shown to be a sensitive measure of orientation of the protein film. On both aluminum and indium-tin-oxide surfaces, XPS indicates that the Ru-Fe axis of the myoglobin derivative is preferentially oriented with the Ru furthest from the substrate.