Nitric oxide as an intermediate in denitrification: evidence from nitrogen-13 isotope exchange

Label exchange studies were used to investigate the role of nitric oxide as an intermediate of denitrification in $\text{Pseudomonas aureofaciens}$ and $\text{Pseudomonas chlororaphis}$. The [¹³N]N from [¹³N]NO₂^? readily exchanged with pools of added, nonlabeled NO, with 54% of the [¹³N]N appearing in a pool of 7.2 × 10^?3 atm NO in $\text{P}\text{.}\text{aureofaciens}$. These results suggest that NO is either an intermediate in the reductive sequence or is in rapid equilibrium with an unidentified intermediate.     

pH-Abhängigkeit der 13C-15N-kopplungskonstanten 15N-hochmarkierter aminosäuren,gewonnen aus algenmassenkulturenpH dependence of 13C-15N coupling constants of highly 15N-enriched amino acid isolated from mass cultivation of algae

The alga Ankistrodesmus braunii was grown with [¹⁵N]nitrate under the optimized conditions of a large-scale mass cultivation. 19.7% of the dried algae were isolated as a mixture of amino acids. The ¹⁵N-labelled amino acids (¹⁵N content up to 98%) were separated by ion exchange chromatography using pyridine acetate gradients. The ¹⁵N content of the analytically pure amino acids was determined by combined gas-liquid chromatography-mass spectrometry of the trifluoroacetylated methylesters and by emission spectroscopy in the ¹⁵N analysator. Using pulse Fourier transform ¹³C nuclear magnetic resonance, the pH dependence of the ¹³C-¹⁵N coupling constants of Asp, Pro, Ser, Glu, Gly, Ala, Val, Ile and Leu was determined in aqueous solutions. Increasing coupling constants were found with pH and decreasing electron density, respectively. The relation of Binsch et al. (Binsch, G., Lambert, J.B., Roberts, B.W. and Roberts, J.D. (1964) J. Am. Chem. Soc. 86, 5564–5570) between the coupling constant and the product of the S-part of the ¹³C and ¹⁵N hybridization $\text{S}{}_{\mathrm{C}}\text{·}\text{S}{}_{\mathrm{N}}\text{= 80 · J (}{}^{13}\text{C}\text{-}{}^{15}\text{X}\text{)}$ fits best in acidic medium. The magnitude of the coupling constants correlates well with the electron densities calculated by Del Re et al. (Del Re, G., Pullman, B. and Yonezawa, T. (1963) Biochim. Biophys. Acta 75, 153–182). The recording of ¹³C nuclear magnetic resonance spectra over the entire pH range revealed no change in the sign of the ¹³C-¹⁵N coupling constants of the amino acids.     

Secondary structure of peptidese: 13. 15N and 13C n.m.r. spectroscopic investigation of the helix stability of poly(l-alanine) in acidic solutions

¹⁵N-enriched poly(l-alanines) of various molecular weights were prepared from $\text{l}\text{-alanine}\text{-N-}\text{carboxyanhydride}$ (l-Ala-NCA) and their helix/coil equilibrium in trifluoroacetic acid (TFA) investigated by means of 40.5 MHz ¹⁵N nuclear magneic resonance (n.m.r.), 22.3 MHz ¹³C n.m.r. and circular dichroism (c.d.) spectra. The ¹⁵N n.m.r. spectra exhibit at least three peaks, and the dependence of their intensities on molecular weight, molecular weight distribution and temperature, as well as dynamic nuclear Overhauser effect (NOE) measurements, indicate that the high-field peak represents the helix fraction. All three spectroscopic methods agree that a helix→coil transition takes place with decreasing concentration. Furthermore, poly(l-alanines) containing d-alanine or glycine in various mole ratios were synthesizsed by copolymerizations of N-carboxyanhydrides (NCAs). The ¹⁵N n.m.r. spetra demonstrate that one d-Ala unit per 100 l-Ala units suffices to affect significantly the helix/coil equilibrium in TFA. In other words, the helix content under equilibrium conditions is highly sensitive to racemization. Furthermore, ¹³ C n.m.r. cross-polarization/magic angle spinning (CP/MAS) spectra demonstrate that the presence of d-Ala units also affects the α-helix content in the solid state.     

5' Cap methylation of homologous poly A(+) RNA by a RNA (guanine-7) methyltransferase from Neurospora crassa.

RNA (guanine-7) methyltransferase, partially purified from $\text{N.}\text{crassa}$ mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both $\text{N.}\text{crassa}$ poly A(+) RNA and reovirus unmethylated mRNA. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated poly A(+) RNA from $\text{N.}\text{crassa}$ yielded the “cap” structures m ⁷G(5′)pppAp and m ⁷G(5′)pppGp in a ratio of 2:1 respectively. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated reovirus mRNA yielded m ⁷G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in $\text{N.}\text{crassa}$ mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. $\text{187}$, 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate.     

The effects of environmental factors on growth and the chemical and biochemical composition of marine diatoms. I. Light and temperature effects

Temperature and light interact to modify the chemical and biochemical composition of a nitrogen-limited marine diatom, Thalassiosira allenii Takano, grown at a constant dilution rate in continuous culture and under a light:dark cycle.The percent of the total ¹⁴C incorporated into protein, polysaccharide and lipid, the N/C ratio and the C/cell varied with temperature in a markedly non-linear manner. The N/cell was negatively correlated to temperature. The Chl $\text{a}\text{C}$ ratio was positively correlated with temperature under saturating light and non-saturating light for temperatures > 25 °C, but was constant under non-saturating light conditions for temperatures < 25 °C.Productivity index (PI) was negatively correlated to temperature under saturating light conditions, but did not vary under low light. In each case, the variation in PI with temperature was governed by the variation in Chl $\text{a}\text{C}$.The dark carbon loss rate was exponentially related to temperature and independent of light. Variation in the percent of the total ¹⁴C incorporated into protein and polysaccharide, the $\text{N}\text{C}$ ratio and C/cell was primarily due to the effects of N-limitation < 20 °C and primarily due to the effects of temperature > 20 °C. Variation in N/cell was primarily due to the effects of temperature over the entire range of temperature studied. Variation in Chl $\text{a}\text{C}$ was caused by the interaction of temperature and light effects.In most cases, temperature and nutrient effects interacted to govern how a particular parameter varied with temperature while light affected the range of values over which the parameter varied.The percent of the total ¹⁴C incorporated into protein exhibited a significant linear relationship with $\text{N}\text{C}$.The dark carbon loss rate, $\text{N}\text{C}$ ratio and Chl $\text{a}\text{C}$ ratio data were used to test the applicability of a model for the physiological adaptation of unicellular algae. The model, with parameters derived from a non-linear least-squares fit of the dark carbon loss rate data, adequately described the $\text{N}\text{C}$ ratio between 15 and 25 °C at 290 and 137 μE · m^?2 · s^?1, but failed to describe the data at 28 °C and at 48 μE · m^?2 · s^?1. The Chl $\text{a}\text{C}$ ratio was adequately described by the model under all light and temperature conditions.     

Resonance raman spectroscopy of chemically modified and isotopically labelled purple membranes. I. A critical examination of the carbon-nitrogen vibrational modes

Resonance Raman spectra of bacteriorhodopsin are compared to the spectra of this protein modified in the following ways: (1) selective deuteration at the C-15 carbon atom of retinal, (2) full deuteration of the retinal, (3) the addition of a conjugated double bond in the β-ionone ring (3-dehydroretinal), (4) full deuteration of the protein and lipid components, (5) ¹⁵N enrichment of the entire membrane and (6) deuteration of the entire membrane (including the retinal). A detailed comparison of the ¹⁵N-enriched membrane and naturally occurring purple membrane from 800 cm^?1 to 1700 cm^?1 reveals that ¹⁵N enrichment affects the frequency of only two vibrational modes. These occur at 1642 cm^?1 and 1620 cm^?1 in naturally occurring purple membrane and at 1628 cm^?1 and 1615 cm^?1 in the ¹⁵N-enriched samples. Therefore, this pair of bands reflects the states of protonation of the Schiff base. However, our data also indicate that neither of these modes are simple, localized $\text{C}\text{=}\textcolor{red}{}\text{?}\text{H}$ or C=N stretching vibrations. In the case of the 1642 cm^?1 band motions of the retinal chain beyond C-15 are not significantly involved. On the other hand, in the 1620 cm^?1 band atomic motions in the isoprenoid chain beyond C-15 are involved.     

Enzymatic formation of 14,15-leukotriene A and C(14)-sulfur-linked peptides

Human leukocytes converted [³H]-(S)-15-HPETE into [³H]-14,15-LTA. Rat basophilic leukemia cells transformed 14,15-LTA into two bioactive C(14)-S-linked peptides, which have been characterized as 15(S)-hydroxy-14(R)-S-glutathionyl-5,8$\text{Z}$,10,12$\text{E}$-icosatetraenoic acid and 15-(S)-hydroxy-14(R)-S-cysteinylglycyl-5,8$\text{Z}$,10,12$\text{E}$-icosatetraenoic acid by comparison with synthetic specimens.     

The role of the lysines in the alkaline heme-linked ionization of ferric cytochrome c.

Phage ?¹⁵ adsorbed at a low temperature (or by short-time incubation) to the outer surface of $\text{Salmonella}\text{anatum}$ gathers on further incubation at a high temperature to a certain region where the inner and outer membranes may join. This was demonstrated by separating the inner and outer membranes of the cells in sucrose gradient after addition of ³⁵S-labeled ?¹⁵ to the cells. Radioactivity adsorbed at 4° was first recovered mainly with the dense outer membrane but disappeared by further incubation at 35° within 5 min. Instead, the radioactivity was recovered with the membrane fraction which had intermediate density. Such phage translocation was not observed when phage ?¹⁵ was added to a $\text{pep}\text{mutant of}\text{S.}\text{anatum}$ to which the phage can adsorb but fail to infect. A host range mutant phage which can infect the $\text{pep}$ mutant migrated to the intermediate dense region.     

Nδ-(phosphonacetyl)-L-ornithine,a potent transition state analogue inhibitor of ornithine carbamoyltransferase

$\text{N}$^δ-(Phosphonacetyl)-L-ornithine, a transition state analogue for the reaction catalyzed by ornithine carbamoyltransferase (EC 2.1.3.3), was synthesized. It strongly inhibited bovine liver ornithine carbamoyltransferase. The inhibition was competitive with respect to carbamoyl-phosphate; the apparent $\text{K}$_m values for carbamoyl-phosphate were 15 μM in 0.05 M $\text{N}$-2-hydroxyethylpiperazine-$\text{N′}$-2-ethanesulfonate (pH 7.2) and 33 μM in 0.1 M Tris-HCl (pH 8.5), and the inhibition constants at pH 7.2 and 8.5 were 7.1 and 4.7 nM, respectively. The inhibition was non-competitive with L-ornithine, the other substrate of the enzyme. This analogue may provide an effective reagent for the elucidation of carbamoyl-phosphate metabolism and its regulation in the liver of ureotelic animals.     

Reversible redox titrations of cytochrome c and cytochrome c oxidase using detergent solubilized electrochemically generated mediator-titrants

The repetitive, $\text{reversible}$ equilibrium redox cycling of cytochrome $\text{c}$, cytochrome $\text{c}$ oxidase, or mixtures thereof has been made possible by the use of the oxidant, ferricinium ion. This ion is electrochemically generated by the use of non-ionic detergent solubilized ferrocene which is apparently incorporated as micelles and readily electron transfers with an electrode. The $\text{ferricinium-ferrocene}$ couple equilibrates rapidly with these heme proteins. Electrochemically generated benzylviologen radical cations are used as the reductant. The E^O′ values for cytochrome $\text{c}$ oxidase at pH 7.0 are 209 ± 15 mv (2e^?) and 340 ± 15 mv (2e^?).     

The effect of silage additives containing formaldehyde on the fermentation of ryegrass ensiled at different dry matter levels and on the nutritive value of direct-cut silage

Ryegrass, harvested at the pre-ear emergence stage of growth, was ensiled in laboratory silos, either fresh (175 g dry matter kg^?1) or wilted to five DM levels ranging from 216–432 g DM kg^?1, with and without additive treatment. The additives used were “Sylade” containing sulphuric acid (15%) and formaldehyde (23%) applied at 4.6 l t^?1 and an “ADD-F” $\text{(85\%}\text{formic acid}\text{)}\text{formalin}$ mixture (7:3 by volume) applied at a similar rate (4.8 l t^?1). An additional treatment included application of the mixture at a constant rate related to the DM content of the ensiled crop (25 l t^?1 DM).In the untreated silages, the water-soluble carbohydrates (WSC) varied, respectively (over the DM range 175–432), from 0–32 g kg^?1 DM compared with 197-6 g kg^?1 DM for the “Sylade” treated silages and 256-50 g kg^?1 DM for the formic acid/formalin silages treated at an additive rate of 4.8 l t^?1. Corresponding ranges of protein N for the control and treatments (expressed as g kg^?1 total N) were 302–447, 624-502 and 620-505, respectively. When the formic acid/formalin additive was applied at a constant level related to the DM content of the crop, although the WSC content decreased with increasing DM (247-158 g kg^?1 DM), the protein N content (612 g kg^?1 total N) remained constant.Grass from the same field was ensiled fresh, treated with “ADD-F” at the rate of 3.4 l t^?1 fresh grass, $\text{“}\text{ADD-F}\text{”}\text{formalin}$ at the rate of 4.8 l t^?1 fresh grass and “Sylade” at the rate of 4.6 l t^?1 fresh grass. The silages were given to Suffolk-cross wether lambs in digestibility and intake trials. Digestibility coefficients of DM and energy of the silage treated with “Sylade” were significantly lower (P < 0.05) than those of the other three silages. The DM intakes of all the silages were high, ranging from 27.7 g kg^?1 live weight for the “Sylade” silage to 30.7 g kg^?1 live weight for the silage treated with $\text{“}\text{ADD-F}\text{”}\text{formalin}$. Live weight gains ranged from 200 g/day for the control silage to 267 g/day for the $\text{“}\text{ADD-F}\text{”}\text{formalin}$ silage.     

Basal and potassium stimulated, calcium dependent, endorphins release from pituitary cells.

Mouse pituitary tumor cells grown in tissue culture release endorphins spontaneously to the culture medium. Depolarization of these cells by incubation with high K⁺ concentration (56 mM) increased 2–3 folds the release of endorphins. The K⁺ evoked release was Ca⁺⁺ dependent by that: $\text{a}$, removal of Ca⁺⁺ ions inhibited 90% of K⁺ stimulated release. $\text{b}$, ethyleneglycol-bis (β-aminoethyl ether) N,N′-tetraacetic acid (EGTA) inhibited release of endorphins in the presence of high K⁺ and Ca⁺⁺. It is suggested that dual regulatory system inhibit and/or stimulate $\text{in-vivo}$ release of endorphins from the pituitary glands.     

Synthesis and antitumor activity of platinum complexes of protonated diamines

Complexes of the formula cis-[Pt(H$\text{N}\text{+}$^～N)(L)Cl₂], where (H$\text{N}\text{+}$^～N) are the protonated diamines including 3-aminoquinuclidine, N-aminopiperidine, piperazine, N-methylpiperazine, 1,1,4-trimethylpiperazine, and N-methyl-1,4-diazabicyclo [2,2,2] octane (N-methyl-dabco) and L = SCN^?, NO₂^?, Br^?, and F^?, were synthesized from the protonated diamine complexes, [Pt(H$\text{N}\text{+}$^～N)Cl₃]. The antitumor activities of the complexes were evaluated in vitro against L1210 murine leukemia cells, and ID₅₀ values for the L-substituted complexes were compared to values of the parent complexes. In each case it was found that replacement of a chloride ion by SCN^?, NO₂^?, Br^?, or F^?, either reduced or completely eliminated antitumor activity. This effect is explained in terms of the trans-directing ability of the ligand, L, compared to chloride. The NO₂-substituted complex of 3- aminoquinuclidine was tested in vivo and found to exhibit little or no antitumor activity.     

Electron paramagnetic resonance of single crystal 15N-nitrosyl-57Fe-myoglobin

Apomyoglobin and ⁵⁷Fe-enriched (86%) hemin have been reconstituted and the product crystallized. Subsequent reaction with ¹⁵NO (98% enriched) gave ⁵⁷Mb¹⁵NO. Epr of the monoclinic crystals shows a 5.2 ± 0.3 splitting within ± 20° of the $\text{a}{}^1$-axis which is attributable to ⁵⁷Fe ($\text{I}\text{=}\text{1}\text{2}$) splitting. The result suggests a 45% spin density at the iron nucleus in MbNO.     

The inhibitor effect of probenecid and structural analogues on organic anions and chloride permeabilities in ox erythrocytes

Probenecid inhibits anion movements (organic anions and chloride) in ox erythrocytes. The I₅₀ is 4 · 10^?5 M. Structural analogues such as carinamide, $\text{p-}\text{carboxybenzene}$ sulfonamide and $\text{p-}\text{carboxy}$$\text{N,N}$ diethyl benzene sulfonamide, which are drugs of the sulfonamide class, were also found to inhibit anion transport. These results reinforce the previously discussed view based on structural considerations, that sulfonamides act on the red cell membrane as competitors of anion transport. It is possible that probenecid and carinamide act in a similar way in the kidney.     

The phosphorylation of troponin B by phosphorylase b kinase in skeletal muscle of mice carrying the phosphorylase b kinase deficiency gene

In skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene there is < 1% of the normal activity to convert phosphorylase $\text{b}$ to $\text{a}$ in the presence of Ca⁺⁺, Mg⁺⁺, and ATP (1). Correspondingly, there is < 1% of the normal activity to phosphorylate phosphorylase $\text{b}$. Nevertheless, under the same conditions, these extracts catalyze the phosphorylation of troponin at a rate 57% of normal. Phosphorylase $\text{b}$ converting activity can be sedimented from skeletal muscle of control mice by centrifugation. This fraction isolated from I strain skeletal muscle extracts phosphorylates troponin at a rate 29–39% of the control. EGTA¹ (15 mM) inhibits troponin phosphorylation by 50–60% in this fraction from both strains. The EGTA inhibition is reversed by 15 mM Ca⁺⁺. Thus the phosphorylase $\text{b}$ kinase in skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene can phosphorylate troponin B, although it shows little or no activity with phosphorylase as a substrate. This observation is consistent with the normal muscle contractility of I strain animals.     

Occurrence of cis-7-tetradecenoic acid in the envelope phospholipids of Escherichia coli K12

We have isolated a tetradecenoic acid from $\text{E}\text{.}\text{coli}$ and have identified this new acid as $\text{cis}$-7-tetradecenoic by its ¹³C nuclear magnetic resonance spectrum. This identification was confirmed by conventional structural studies. The acid is a component of the phospholipids of $\text{E}\text{.}\text{coli}$ and comprises about 15% of the total phospholipid unsaturated fatty acid.     

Conversion of N6-isopentenyladenine to zeatin by Actinidia tissues

Tissue cultures grown from stem explants of three $\text{Actinidia}$ species and a hybrid species rapidly converted N6-isopentenyladenine (i⁶Ade) to zeatin (io⁶Ade), a potent hydroxylated cytokinin. Within 24 h on 50 uM i⁶Ade, callus tissues of $\text{A.}\text{chinensis}\text{×}\text{arguta}$ accumulated 83 ± 6 nmol/g io⁶Ade which was purified using HPLC and identified by its characteristic UV and mass spectra. Activity converting i⁶Ade to io⁶Ade was also demonstrated in stem segments from intact plants where it was low in the tip (3 cm), highest in the region corresponding to rapid leaf growth and very low in the mature stem. Root segments converted i⁶Ade to io⁶Ade almost as rapidly as the most active region of the stem while leaf petioles produced little io⁶Ade. Fruits of $\text{A.}\text{arguta}$ and $\text{A.}\text{chinensis}$ produced little or no io⁶Ade, respectively.     

Temperature dependence of active K+ transport in cation dimorphic sheep erythrocytes

Arrhenius diagrams of K⁺ pump fluxes measured between 15°C and 41°C were discontinuous in high K⁺ but not in low K⁺ sheep red cells. Exposure of low K⁺ cells to anti-L caused a bimodal temperature response of K⁺ pump flux with a transition temperature, $\text{T}\text{c}$, similar to that found in high K⁺ cells but with comparatively higher activation energies above $\text{T}\text{c}$.     

Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugates of cortisol and cortisone.

Superior antitumor activity of 1-β-D-arabinofuranosylcytosine (ara-C) conjugates of prednisolone and prednisone against L1210 leukemic mice, based on ara-C content, has encouraged us to synthesize 5′-(cortisone-21-phosphoryl)-1-β-D-arabinofuranosylcytosine (I) and 5′-(cortisone-21-phosphoryl)-1-β-d-arabinofuranosylcytosine (II) by condensation of N⁴,2′,3′-triacetyl-1-β-d-arabinofuranosylcytosine 5′-monophosphate with cortisol and cortisone in the presence of N,N′-dicyclohexylcarbodiimide at room temperature followed by removing the acetyl groups in 2 N methanolic ammonia in 20% yield. The conjugates I and II inhibited the $\text{in}\text{vitro}$ growth of L1210 by 50% (ED₅₀) at 0.25 μM and 0.07 μM, respectively, while ara-C showed ED₅₀ 0.1 μM. However, the conjugates I and II exhibited 287% and 238% of $\text{T}\text{C}$ at 50 mg/kg/day × 5 doses against L1210 leukemic mice, respectively, while ara-C at 25 mg and 50 mg/kg/day × 5 gave the respective 127% and 110% of $\text{T}\text{C}$.