Gas chromatographic determination of total amino acids and protein in sediments using the ninhydrin-CO2 reaction

A sensitive modification of the ninhydrin-CO₂ method involving the gas chromatographic determination of the total protein and amino acid content of sediment is described. The method gives a linear response over the amino acid concentration range 10^–5 M to 4 × 10^–2 M. It can be used for whole sediment, hydrolysates and interstitial water. The performance of the method is compared with the fluorescamine method for primary amines.     

Cycloheximide inhibitation on hypoxanthine transport in cultured cells

Cycloheximide preincubation inhibits hypoxanthine uptake into the acid-soluble fractions of cultured rat hepatoma cells (MH₁C₁) and human skin epithelial cells (NCTC 2544, HE cells) in a time- and dose-dependent manner 50% inhibition is seen after 4 h preincubation with 10^?4 M cycloheximide of MH₁C₁ cells and after 2.5 h of HE cells. Adenine uptake is much less affected, after 10 h preincubation with 10^?4 M cycloheximide it was reduced to 83% and 67% of controls in MH₁C₁ cells and HE cells respectively. Cycloheximide inhibits hypoxanthine uptake in a dose-dependent manner above 10^?7 M, with 50% inhibition in MH₁C₁ cells at 4 · 10^?7 M after 12 h preincubation and at 10^-6 M in HE cells after 6 h preincubation. Puromycin mimics the action of cycloheximide. The inhibition of hypoxanthine uptke is not caused by reduction of the activity of hypoxanthine phosphoribosyltransferase in the two cell lines. 10^?4 M cycloheximide preincubation for 10 h does not significantly reduce the uptake of the two non-metabolizable amino acids α-aminoisobutyric acid or 1-aminocyclopentane-1-carboxylic acid (cycloleucine). It is suggested that cycloheximide inhibits the synthesis of a rapidly turning over the protein involved in hypoxanthine transport.     

Copper nanocluster‐enhanced luminol chemiluminescence for high‐selectivity sensing of tryptophan and phenylalanine

A remarkable method for the highly sensitive detection of phenylalanine and tryptophan based on a chemiluminescence (CL) assay was reported. It was found that fluorescent copper nanoclusters capped with cysteine (Cys‐CuNCs) strongly enhance the weak CL signal resulting from the reaction between luminol and H₂O₂. Of the amino acids tested, phenylalanine and tryptophan could enhance the above CL system sensitively. Under optimum conditions, this method was satisfactorily described by a linear calibration curve over a range of 1.0 × 10^?6 to 2.7 × 10^?5 M for phenylalanine and 1.0 × 10^?7 to 3.0 × 10^?5 M for tryptophan, respectively. The effect of various parameters such as Cys‐CuNC concentration, H₂O₂ concentration and pH on the intensity of the CL system were also studied. The main experimental advantage of the proposed method was its selectivity for two amino acids compared with others. To evaluate the applicability of the method to the analysis of a real biological sample it was used to determine tryptophan and phenylalanine in human serum and remarkable results were obtained.     

Rapid stimulatory effect of thyroxine on plasma membrane transport systems: Calcium uptake and neutral amino acid accumulation in immature rat testis

Although the biological effects of thyroid hormones are mediated by nuclear receptors (genomic mechanisms), interactions with receptors associated with the plasma membrane (non-genomic mechanisms) of target cells are not clear. In this study we investigated the rapid stimulatory effect of thyroxine (T₄) on ⁴⁵Ca²⁺ uptake as well as ionic currents and intracellular messengers involved in the stimulatory action of T₄ in amino acid accumulation in immature rat testes. Results indicated that 10^?9 M or 10^?6 M T₄ was able to increase immediately ⁴⁵Ca²⁺ uptake after 60 s of hormone exposure. These results indicate for the first time that voltage-dependent Ca²⁺ channels and ATP-dependent K⁺ channels can be seen as a set-point in the stimulatory effect of T₄ on amino acid accumulation. Apamin-sensitive small-conductance Ca²⁺-activated K⁺ channels (SK_Ca) and chloride channels were shown to be partially involved in this mechanism. The amino acid accumulation triggered by the PKC pathway suggests a functional link between different ion channel activities and the stimulatory effect of T₄ on amino acid accumulation. In conclusion, we show in this study a rapid and stimulatory effect of T₄ on calcium uptake and on amino acid accumulation, both events initiated at the plasma membrane, which strongly characterizes a non-genomic effect of T₄ in immature rat testes.     

Bone resorption and prostaglandin production by mouse calvaria in vitro: Response to exogenous prostaglandins and their precursor fatty acids

Mouse calvaria were maintained in organ culture for 96 h and endogenous prostaglandin production and active bone resorption (⁴⁵ Ca release) measured. After a lag phase of 12 h, active resorption increased over the 96 h period. The amounts of prostaglandins released into the culture medium (measured by radioimmunoassay) were highest in the first 24 h of culture. Unless these were removed by preculturing for 24 h, or suppressed by indomethacin, no response to exogenous PGE₂, PGF_2α or prostaglandin precursors could be demonstrated. Bone resorption was stimulated after preculture by both PGE₂ and PGF_2α in a dose-dependent manner (10^?18M – 10^?5M), with PGE₂ being the more potent. Collagen synthesis was unaffected by PGF_2α, whereas PGE₂ (10^?5M) had an inhibitory effect. Eicosatrienoic acid did not stimulate bone resorption at lower concentrations (10^?7M – 10^?5M_, but was inhibitory at 10^?4M. Arachidonic acid also inhibited resorption at 10^?4M, but at lower concentrations (10^?7M – 10^?5M0 increased active resorption. This was concomitant with a rise in PGE₂ and PGF_2α levels, PGE₂ production being significantly higher than PGF_2α. The effects of PGE₂ (10^?8M) and PGF_2α (10^∞M appeared additive: there was no evidence of synergistic or antagonistic effects when varying ratios of PGE₂ : PGF_2α were employed.     

Crystallization and amino acid composition of cathepsin B from rat liver lysosomes

Cathepsin B (EC 3.4.22.1) from rat liver was crystallized and its amino acid composition was determined. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by ultracentrifugical analysis. Its S_{20, W} value was 2.5 S and its molecular weight was calculated to be 24,000 from the result of sedimentation equilibrium analysis. Amino acid analysis showed that it contained glucosamine and galactosamine. The activity of the protease was maximal at pH 6.0 with α-N-benzoyl-DL-arginine p-nitroanilide as substrate. The apparent Kms for α-N-benzoyl-DL-arginine p-nitroanilide and α-N-benzoyl-DL-arginine-2-naphthylamide were 1.4 × 10^?2 M and 2.0 × 10^?3 M, respectively     

Combined effects of water column nitrate enrichment, sediment type and irradiance on growth and foliar nutrient concentrations of Potamogeton alpinus

1. High water column NO₃^? concentrations, low light availability and anoxic, muddy sediments are hypothesised to be key factors hampering growth of rooted submerged plants in shallow, eutrophic fresh water systems. In this study, the relative roles and interacting effects of these potential stressors on survival, growth, allocation of biomass and foliar nutrient concentrations of Potamogeton alpinus were determined in a mesocosm experiment using contrasting values of each factor (500 versus 0 μmol L^?1 NO₃^?; low irradiance, corresponding to the eutrophic environment, versus ambient irradiance; and muddy versus sandy sediment). 2. Low irradiance, high NO₃^? and sandy sediment led to reduced growth. In a muddy sediment, plants had lower root : shoot ratios than in a sandy sediment. 3. Growth at high NO₃^? and on the sandy sediment resulted in lower foliar N and C concentrations than in the contrasting treatments. The C : N ratio was higher at high NO₃^? and on the sandy sediment. Foliar P was higher on the muddy than on the sandy sediment but was not affected by irradiance or NO₃^?. The N : P ratio was lowest at high NO₃^? on the sandy sediment. 4. Total foliar free amino acid concentration was lowest on sand, low irradiance and high NO₃^?. Total free amino acid concentration and growth were not correlated. 5. Turbidity and ortho‐PO₄^3? concentration of the water layer were lower at high water column NO₃^? indicating that the growth reduction was not associated with increased algal growth but that physiological mechanisms were involved. 6. We conclude that high water column NO₃^? concentrations can significantly reduce the growth of ammonium preferring rooted submerged species such as P. alpinus, particularly on sediments with a relatively low nutrient availability. Further experiments are needed to assess potential negative effects on other species and to further elucidate the underlying physiological mechanisms.     

Inhibition of the neutrophil oxidative response to a chemotactic peptide by inhibitors of arachidonic acid oxygenation

N-formylmethionylphenylalanine stimulates a short burst of antimycin A-insensitive O₂ uptake, O₂^? production and hexosemonophosphate shunt oxidation of glucose by guinea pig peritoneal neutrophils. The stimulated oxidative metabolism, as well as release of lysosomal enzymes ± cytochalasin B, are inhibited by 5,8,11,14-eicosatetraynoic acid (ID₅₀ 1.5 × 10^?5 M). High concentrations of indomethacin inhibit the peptide-stimulated oxidations (ID₅₀ 1.6 × 10^?4 M) while acetylsalicylic acid (2.5 × 10^?3 M) does not. Digitonin-stimulated oxidative metabolism and enzyme release are not inhibited by 5,8,11,14-eicosatetraynoic acid or indomethacin at concentrations that depress effects of the N-formylated peptide.     

Effects of estradiol and progesterone on uterine prostaglandin levels in the pregnant rat

Prostaglandin D₂ was found to be a potent inhibitor of B-16 melanoma cell replication $\text{in vitro}$. The inhibition was dose-dependent between 3×10^?9M and 3×10^?6M (IC_50～ 0.3 μM after 6 days). On a molar basis, PGD₂ was a better inhibitor than PGA₂ or 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) and in higher concentrations (10^?6?10^?7M), comparable to retinoic acid. In higher concentrations, PGD₂ inhibited DNA, RNA and protein synthesis. The B-16 melanoma cell line which we used synthesized arachidonic acid metabolites which comigrated with PGA₂, PGD₂, PGE₂ and PGF_2α on a thin layer chromatography system.     

Catalytic properties of three lactate dehydrogenases from potato tubers (Solanum tuberosum)

Two l-lactate dehydrogenase isoenzymes and one dl-lactate dehydrogenase could be separated from potato tubers by polyacrylamide-gel electrophoresis. The enzymes are specific for lactate, while β-hydroxybutyric acid, glycolic acid, and glyoxylic acid are not oxidized. Their pH optima are pH 6.9 for the oxidation and 8.0 for the reduction reaction.The K_m values for l-lactate for the two isoenzymes are 2.00 × 10^?2 and 1.82 × 10^?2, m. In the reverse reaction the affinities for pyruvate are 3.24 × 10^?4 and 3.34 × 10^?4, m. Both enzymes have similar affinities for NAD and NADH (3.00 × 10^?4; 4.00 × 10^?4, and 8.35 × 10^?4; 5.25 × 10^?4, m).The dl-lactate oxidoreductase may transfer electrons either to NAD or N-methyl-phenazinemethosulfate. The K_m values of this enzyme for l-lactate are 4.5 × 10^?2, m and for d-lactate 3.34 × 10^?2, m. Its affinity for pyruvate is 4.75 × 10^?4, m. The enzyme is inhibited by excess NAD (K_m = 1.54 × 10^?4, M) and has an affinity toward NADH (K_m = 5.00 × 10^?3, M) which is about one tenth of that of the two isoenzymes of l-lactate dehydrogenase.     

IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins

The formaldehyde method was used to examine the interaction of PGE₁ with morphine, β-endorphin and Met-enkephalin on rat mast cells by their effects on IgE-mediated ¹⁴C-serotonin release. PGE₁ (2×10^?8?2×10^?5 M) caused a dose-related inhibition of the mediator release 1 min after an antigen challenge, and morphine (3×10^?7?3×10^?5 M) reversed this PGE₁ effect dose-dependently and stereospecifically; naloxone (2×10^?4 M) antagonized this action of morphine. β-Endorphin (3×10^?7?10^?5 M) and Met-enkephalin (3×10^?6?10^?4 M) mimicked this morphine action dose-dependently and were antagonized by naloxone (2×10^?4 M). These results suggest that morphine and endorphins modulate immunological mediator release from rat mast cells through opioid receptors.     

Antirestriction activity of the monomeric and dimeric forms of T7 Ocr

The Ocr antirestriction protein, which is encoded by bacteriophage T7 0.3 (ocr), specifically inhibits type I restriction-modification enzymes. Ocr belongs to a family of DNA-mimicking proteins. Native Ocr forms homodimers both in solution and in crystal. Ocr mutants with two amino acid substitutions (Orc F53D A57E and Ocr F53R V77D) were constructed to occur as monomers in solution. The dissociation constant K _d for the Ocr complex with EcoKI (R₂M₂S) proved to differ by three orders of magnitude between the (Ocr)₂ dimer and Ocr F53D A57E and Ocr F53R V77D monomers (10^?10 M vs. 10^?7 M). Antimodification activity was substantially lower in the Ocr monomers. The dimeric form found to be essential for high inhibitory activity of Ocr.     

KINETICS OF AMINO ACID INFLUX INTO NITELLA FLEXILIS1

The uptake of amino acids by Nitella flexilis has been investigated. Influx of glycine, alanine, and valine appears to be a diffusive process. Influx ranged from 0.14 to 0.06 and 0.04 pmoles/(cm)(sec), respectively. Aspartic acid uptake is an active transport mechanism. The V_max is 2.8 pmoles/(cm)(sec); the transport constant (Michaelis constant) K_m, 7.8 × 10^?3 M. The uptake of arginine is apparently due to 2 transport systems, one with a V_max and K_m of 3.1 pmoles/(cm)(sec) and 3.2 × 10^?3M, respectively. The second system has a V_max of 1.4 pmoles/(cm)(sec) and a K_m of 2.1 × 10^?4 M. The possibility that the second system is diffusive has been considered.     

Isolation and structure of a novel peptide inhibitor of HIV-1 integrase from marine polychaetes

A homogeneous peptide with a mass of 683 Da which inhibits HIV-1 integrase with IC₅₀ 3 × 10^?5 M was separated from aqueous extracts of a marine worm Eunicidae sp. by multistage chromatography purification. The Asp-Leu-Hse-His-Ala-Gln structure was proposed for this peptide according to amino acid analysis, automated amino acid Edman sequences, and TLC with witness homoserine and MS/MS fragmentation. The proposed structure is the first example of a natural peptide containing an amino acid homoserine residue.     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

Denitrification and nitrous oxide effluxes in boreal, eutrophic river sediments under increasing nitrate load: a laboratory microcosm study

Intact sediment cores from rivers of the Bothnian Bay (Baltic Sea) were studied for denitrification based on benthic fluxes of molecular nitrogen (N₂) and nitrous oxide (N₂O) in a temperature controlled continuous water flow laboratory microcosm under 10, 30, 100, and 300 μM of ¹⁵N enriched nitrate (NO₃ ^?, ~98 at. %). Effluxes of both N₂ and N₂O from sediment to the overlying water increased with increasing NO₃ ^? load. Although the ratio of N₂O to N₂ increased with increasing NO₃ ^? load, it remained below 0.04, N₂ always being the main product. At the NO₃ ^? concentrations most frequently found in the studied river water (10–100 μM), up to 8% of the NO₃ ^? was removed in denitrification, whereas with the highest concentration (300 μM), the removal by denitrification was less than 2%. However, overall up to 42% of the NO₃ ^? was removed by mechanisms other than denitrification. As the microbial activity was simultaneously enhanced by the NO₃ ^? load, shown as increased oxygen consumption and dissolved inorganic carbom efflux, it is likely that a majority of the NO₃ ^? was assimilated by microbes during their growth. The ¹⁵N content in ammonium (NH₄ ⁺) in the efflux was low, suggesting that reduction of NO₃ ^? to NH₄ ⁺ was not the reason for the NO₃ ^? removal. This study provides the first published information on denitrification and N₂O fluxes and their regulation by NO₃ ^? load in eutrophic high latitude rivers.     

The presence of discrete receptors for prostaglandin F2 alpha in the cell membranes of bovine corpora lutea.

The cell membranes isolated from bovine corpora lutea bound ³H-prostaglandin (PG) F₂α with high affinity and specificity. The specific binding of ³H-PGF₂α was detectable at 10^?10M added ³H-PGF₂α and reached saturation at 10^?7M to 10^?6M. Unlabeled PGF₂α, as low as 10^?9M, inhibited the binding of ³H-PGF₂α with complete inhibition occurring at 10^?6M. The Scatchard analysis of equilibrium binding data revealed that the PGF₂α receptors are heterogeneous: Kd₁?5.1 × 10^?9M, n?289 fmoles/mg protein; Kd₂?1.8 × 10^?8M, n?780 fmoles/mg protein. The relative affinities of various other PGs for binding to PGF₂α receptors were (PGF₂α?100%): PGF₁α?17.5; PGE₁?0.8; PGE₂?22.4; PGA₁?0.007; PGB₁?0.01. The specificity and affinity of ³H-PGF₂α binding is consistent with the possibility that this receptor interaction may reflect an initial event in the action of PGF₂α as a luteolytic agent.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^?8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^?8 M and 7×10^?7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^?8 M and 6×10^?8 M, respectively.     

Biochemical characterisation of a Kunitz-type inhibitor from Tamarindus indica L. seeds and its efficacy in reducing plasma leptin in an experimental model of obesity

A trypsin inhibitor isolated from tamarind seed (TTI) has satietogenic effects in animals, increasing the cholecystokinin (CCK) in eutrophy and reducing leptin in obesity. We purified TTI (pTTI), characterised, and observed its effect upon CCK and leptin in obese Wistar rats. By HPLC, and after amplification of resolution, two protein fractions were observed: Fr1 and Fr2, with average mass of [M?+?14H]⁺?=?19,594,690?Da and [M?+?13H]⁺?=?19,578,266?Da, respectively. The protein fractions showed 54 and 53 amino acid residues with the same sequence. pTTI presented resistance to temperature and pH variations; IC₅₀ was 2.7?×?10^?10?mol.L^?1 and Ki was 2.9?×?10^?11?mol.L^?1. The 2-DE revealed spots with isoelectric points between pH 5 and 6, and one near pH 8. pTTI action on leptin decrease was confirmed. We conclude that pTTI is a Kunitz trypsin inhibitor with possible biotechnological health-related application.     

Differential in vitro inhibition of polyphenoloxidase from a wild edible mushroom Lactarius salmonicolor

The polyphenol oxidase (LsPPO) from a wild edible mushroom Lactarius salmonicolor was purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. At the optimum pH and temperature, the K_M and V_Max values of LsPPO towards catechol, 4-methylcatechol and pyrogallol were determined as 0.025 M & 0.748 EU/mL, 1.809 × 10^{? 3} M & 0.723 EU/mL and 9.465 × 10^{? 3} M & 0.722 EU/mL, respectively.

Optimum pH and temperature values of LsPPO for the three substrates above ranged between the pH 4.5–11.0 and 5–50°C. Enzyme activity decreased due to heat denaturation with increasing temperature. Effects of a variety of classical PPO inhibitors were investigated opon the activity of LsPPO using catechol as the substrate. IC₅₀ values for glutathione, p-aminobenzenesulfonamide, L-cysteine, L-tyrosine, oxalic acid, β-mercaptoethanol and syringic acid were determined as 9.1 × 10^{? 4}, 2.3 × 10^{? 4} M, 1.5 × 10^{? 4} M, 3.8 × 10^{? 7} M, 1.2 × 10^{? 4} M, 4.9 × 10^{? 4} M, and 4 × 10^{? 4} M respectively. Thus L-tyrosine was by far the most effective inhibitor. Interestingly, sulfosalicylic acid behaved as an activator of LsPPO in this study.