Prebiotic nucleotide synthesis-demonstration of a geologically plausible pathway

Mineral phosphate (apatite) is activated for the synthesis of nucleotides when dilute solutions containing nucleoside and ammonium oxalate are evaporated in its presence. A natural, igneous flourapatite was found to be even more effective in nucleotide synthesis than the more soluble hydroxylapatite. The phosphorylation is considerably more efficient if urea or cyanamide is also present. Hydrolysis of solutions of cyanogen to form oxalate and urea among other products is a spontaneous process that provides a geologically plausible model for nucleotide synthesis on the primitive earth.     

Possible role of hydrogen cyanide in chemical evolution. The oligomerization and condensation of hydrogen cyanide

The rate of cyanide oligomerization is independent of the presence of added nucleophiles such as azide, monomethylamine or triethylamine and is dependent only on the pH of the reaction mixture. The products formed, with the exception of urea, are also independent of the nucleophiles used to initiate reaction. In the presence of monomethylamine, monomethylurea is the main neutral product instead of urea, suggesting the intermediacy of cyanate. Evidence is presented which suggests that cyanogen may be the precurser to both cyanate and oxalic acid in the cyanide Oligomerization.     

Transformation of vanadinite [Pb5(VO4)3Cl] by fungi

Saprotrophic fungi were investigated for their bioweathering effects on the vanadium‐ and lead‐containing insoluble apatite group mineral, vanadinite [Pb₅(VO₄)₃Cl]. Despite the insolubility of vanadinite, fungi exerted both biochemical and biophysical effects on the mineral including etching, penetration and formation of new biominerals. Lead oxalate was precipitated by Aspergillus niger during bioleaching of natural and synthetic vanadinite. Some calcium oxalate monohydrate (whewellite) was formed with natural vanadinite because of the presence of associated ankerite [Ca(Fe²⁺,Mg)(CO₃)₂]. Aspergillus niger also precipitated lead oxalate during growth in the presence of lead carbonate, vanadium(V) oxide and ammonium metavanadate, while abiotic tests confirmed the efficacy of oxalic acid in solubilizing vanadinite and precipitating lead as oxalate. Geochemical modelling confirmed the complexity of vanadium speciation, and the significant effect of oxalate. Oxalate–vanadium complexes markedly reduced the vanadinite stability field, with cationic lead(II) and lead oxalate also occurring. In all treatments and geochemical simulations, no other lead vanadate, or vanadium minerals were detected. This research highlights the importance of oxalate in vanadinite bioweathering and suggests a general fungal transformation of lead‐containing apatite group minerals (e.g. vanadinite, pyromorphite, mimetite) by this mechanism. The findings are also relevant to remedial treatments for lead/vanadium contamination, and novel approaches for vanadium recovery.     

Yersinia pseudotuberculosis nucleoside-kinase

Enzyme capable of catalyzing the phosphorylation of thymidine and uridine was isolated from Y. pseudotuberculosis cells by fractionation with the use of ammonium sulfate, ion exchange and affinity chromatography. The degree of purification of thymidine- and uridine-kinase was approximately 350 times, and at all stages of isolation the activity of both nucleoside-kinases was detected in the same peaks. The purified enzyme was capable of the phosphorylation of thymidine and uridine at temperatures of 8-10 degrees C to 50 degrees C and exhibited the maximum enzymatic activity at pH 8-8.5 and 45 degrees C in the presence of 0.5-1.0 mM MgCl2 and 2 mM ATP. The enzyme was found to have no strict substrate specificity and transferred the phosphate group from ATP to radiolabeled thymidine, uridine and desoxycytidine with different effectiveness, but did not use thymidine-monophosphate as phosphate acceptor.     

Method for purification of large cyanogen bromide peptides by carboxymethyl cellulose chromatography in 8 m urea: Application to the purification of cyanogen bromide peptides from staphylcoccal enterotoxin A,phosphoglycerate kinase,and glucose-6-phosphate dehydrogenase

A method for purification of large cyanogen bromide peptides from proteins by means of carboxymethyl cellulose chromatography in the presence of 8 m urea is described. Chromatography of a number of large cyanogen bromide peptides which could not be separated by gel filtration showed that the resolution of the system was sufficient to enable large cyanogen bromide peptides to be separated from one another. The use of this method to purify cyanogen bromide peptides of a protein as a first step is also discussed.     

Effect of trickle fertigation with three forms of nitrogen on soil pH,levels of extractable nutrients below the emitter and plant growth

The effects of application of nitrogen as calcium nitrate, urea or ammonium sulphate at two rates through the trickle irrigation system on pH and nutrient status of the wetted volume of soil below the emitters and on growth and nutrition of courgette (zucchini) plants (Cucurbita pepo L.) was investigated. Soil acidification, caused by nitrification, occurred to a large extent in the volume of soil immediately below the emitters in the urea and ammonium sulphate treatments. Acidification was greater at the high rate of N addition and more pronounced with ammonium sulphate than urea. A significant amount of applied urea appeared to move through the soil as urea and consequently, at the same rate of N addition, levels of ammonium were lower directly below the emitter and those of nitrate were higher further away from the emitters for the urea than ammonium sulphate treatments. Soil acidification below the emitters resulted in significant decreases in levels of exchangeable Ca, Mg and K and increases in levels of exchangeable Al, EDTA-extractable Fe, Mn, Zn and Cu and bicarbonate-extractable P. Vegetative growth and harvestable yields of courgettes were increased by both irrigation and nitrogen applications. Vegetative growth was generally greater at the low rate of N addition than at the high one and generally followed the order calcium nitrate > urea > ammonium sulphate. However, fruit yields followed the order urea > ammonium sulphate > calcium nitrate and were larger at the high rate of N for urea and ammonium sulphate treatments and unaffected by rate for the calcium nitrate treatments. It is suggested that with fertigation, the form of applied N can have significant physiological effects of plant growth and yields because N may be applied into the root zone on numerous occasions during the growing season.     

Effect of Microorganisms and Microbial Metabolites on Apatite Dissolution

The dissolution rate of apatite was determined in batch reactors in organic acid solutions and in microbial cultures. Inoculum for the cultures was from biotite plus apatite crystals from a granite weathering profile in South Eastern Australia. In both the biotic and the abiotic experiments, etching of the apatite surface leads to the formation of elongated spires parallel to the c axis. Apatite dissolution rates in the inorganic, acetate, and oxalate solutions increase as pH decreases from approximately 10 -11 mol/m -2 · s -1 at initial pH 5.5 to 10 -7 mol/m -2 · s -1 at initial pH 2. Under mildly acidic to near neutral pH conditions, both oxalate and acetate increased apatite dissolution by up to an order of magnitude compared to the inorganic conditions. Acetate catalyzed the reaction by forming complexes with Ca, either in solution or at the mineral surfaces. Oxalate forms complexes with Ca as well, and can also affect reaction rates and stoichiometry by forming Ca-oxalate precipitates, thus affecting solution saturation states. In all abiotic experiments, net phosphate release to solution approaches zero even when solutions are apparently undersaturated by several orders of magnitude with respect to the solubility of an ideal fluoroapatite mineral. In the microbial experiments, two enrichment cultures increased both apatite and biotite dissolution by producing organic acids, primarily pyruvate, fermentation products, and oxalate, and by lowering bulk solution pH to between 3 and 5. However, the microorganisms were also able to increase phosphate release from apatite (by two orders of magnitude) without lowering bulk solution pH by producing pyruvate and other compounds.     

不同因子对荞麦中草酸含量的影响

用不同化合物从根部喂养麦幼苗,测定其根叶中草酸含量的变化。结果表明：异柠檬酸、抗坏血酸及其前体物均可不同程度地降低荞麦根叶中草酸含量;而乙醇酸与乙醛酸则显著提高其草酸含量,表明荞麦叶片草酸合成主要来自乙醇酸途径,而非来自抗坏血酸等途径。水培条件下,以铵态氮或尿素等作唯一氮源时,荞麦中草酸含量远低于以硝态氮培养的;将谷氨酸或丝氨酸加到含硝态氮培养液中也能显著降低其草酸含量,不同氮素影响荞麦草酸含量可能与乙醇酸途径有关。     

Fluoride ion catalyzed alkylation of nucleic acid derivatives using trialkyl phosphates, dialkyl sulfates and alkyl methanesulfonates.

Trimethyl phosphate, dimethyl and diethyl sulfate and methyl and ethyl methanesulfonate all give high yields of alkylation on purines and pyrimidines in the presence of tetrabutylammonium fluoride. Trimethyl phosphate produces near quantitative yields of diesters of nucleic acids but gives virtually no triester formation. The alkyl sulfates produce very high yields of triesters of nucleic acids including cyclic phosphates while the alkyl methanesulfonates are intermediate in reactivity. It was observed that in the absence of fluoride ion the dialkylsulfates gave reasonable yields of thymidine monosulfates.     

Determination of phosphorylation levels of tyrosine hydroxylase by electrospray mass spectrometry

A novel approach has been developed to quantify the extent of phosphorylation of tyrosine hydroxylase (TH). The strategy consists of a chemical cleavage and characterization of the products using electrospray mass spectrometry (ESMS). The chemical cleavage involves selective hydrolysis of the aspartyl-peptide bond. Of the peptides formed, an 8-kDa NH2-terminus fragment is found to accurately duplicate the phosphorylation of TH using standard mixtures of TH-P/TH. The calibration yields a straight line with an R2 of 0.996, which is valid within the 10-90% range. The ESMS protocol has been used to determine the extent of phosphorylation of TH in the presence of CaM-PKII. The experimental conditions were designed to produce low levels of phosphorylation. Nevertheless, the ESMS analysis yielded single, double, and nonphosphorylation forms of TH. With respect to in vivo measurements, this ESMS protocol may be a generic procedure for determining the extent of phosphorylation of proteins.     

The solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptides

Abnormal fragmentation during cyanogen bromide polypeptide cleavage rarely occurs, although parallel side reactions are known to typically accompany normal cleavage. We have observed that cyanogen bromide cleavage of highly hydrophobic fusion proteins utilized for production of recombinant peptides results in almost complete abolishment of the expected reaction products when the reaction is carried out in 70% trifluoroacetic acid. On the basis of mass spectrometric analysis of the reaction products, we have identified a number of fragments whose origin can be attributed to incomplete fragmentation of the fusion protein, and to unspecific degradation affecting the carrier protein. Substituting the solvent in the reaction media with 70% formic acid or with a matrix composed of 6M guanidinium hydrochloride in 0.1M HCl, however, was found to alleviate polypeptide cleavage. We have attributed the poor yields of the CNBr cleavage carried out in 70% TFA to the increased hydrophobicity of our particular fusion proteins, and to the poor solubilizing ability of this reaction medium. We propose the utilization of chaotropic agents in the presence of diluted acids as the preferred cyanogen bromide cleavage medium of fusion proteins in order to maximize cleavage efficiency of hydrophobic sequences and to prevent deleterious degradation and structural modifications of the target peptides.     

Oxalate Accumulation as Regulated by Nitrogen Forms and Its Relationship to Photosynthesis in Rice (Oryza sativa L.)

Four-leaf rice seedlings (Oryza sativa L.), which had been cultivated in Kimura B complete nutrient solution, were treated with two nitrogen forms by replacing the nitrogen element in the complete solution with sole nitrate or ammonium (2.86 mmol/L). Nitrate-N nutrition tended to increase oxalate content in all parts of the plant, including the leaves, stems, roots, and root exudates, whereas ammonium had the opposite effect. Consequently, marked differences in oxalate content were observed between the two treatments throughout the time tested (0--12 d), with maximal differences of approximately 12-fold at 6 d after treatment. Photosynthetic/respiratory parameters were examined over time simultaneously with changes in oxalate content. Net photosynthetic rate, chlorophyll fluorescence parameters (i.e. maximal photochemical efficiency (Fv/Fm) and photochemical quantum yields of photosystem (PS)Ⅱ (φ PSⅡ)), and respiratory rate were not significantly different between plants treated with the two nitrogen forms, although ammonium-fed plants had apparently higher leaf chlorophyll content than nitrate-fed plants. Leaf glucose content was altered little, but the content of fructose, sucrose, and total soluble sugar was significantly higher in the leaves of ammonium-fed plants than nitrate-fed plants, The results indicate that nitrate/ammonium may serve as efficient regulators of oxalate accumulation owing to regulation of metabolism in rice leaves rather than oxalate downward transfer and root excretion, and that photosynthetic metabolism is not directly correlated with the regulation of oxalate accumulation in rice plants.     

Oxalate Accumulation as Regulated by Nitrogen Forms and Its Relationship to Photosynthesis in Rice （Oryza sativa L.）

Four-leaf rice seedlings (Oryza sativa L.), which had been cultivated in Kimura B complete nutrient solution, were treated with two nitrogen forms by replacing the nitrogen element in the complete solution with sole nitrate or ammonium (2.86 mmol/L). Nitrate-N nutrition tended to increase oxalate content in all parts of the plant, including the leaves, stems, roots, and root exudates, whereas ammonium had the opposite effect. Consequently, marked differences in oxalate content were observed between the two treatments throughout the time tested (0-12 d), with maximal differences of approximately 12-fold at 6d after treatment. Photosynthetic/respiratory parameters were examined over time simultaneously with changes in oxalate content. Net photosynthetic rate, chlorophyll fluorescence parameters (i.e. maximal photochemical efficiency (Fv/Fm) and photochemical quantum yields of photosystem (PS)II (ΦPSⅡ)), and respiratory rate were not significantly different between plants treated with the two nitrogen forms, although ammonium-fed plants had apparently higher leaf chlorophyll content than nitrate-fed plants. Leaf glucose content was altered little, but the content of fructose, sucrose, and total soluble sugar was significantly higher in the leaves of ammonium-fed plants than nitrate-fed plants. The results indicate that nitrate/ammonium may serve as efficient regulators of oxalate accumulation owing to regulation of metabolism in rice leaves rather than oxalate downward transfer and root excretion, and that photosynthetic metabolism is not directly correlated with the regulation of oxalate accumulation in rice plants.     

Models of prebiological phosphorylation

The hypothesis that contemporary metabolic pathways evolved from analogous chemical reaction sequences on the primitive Earth leads to a reexamination of models of prebiological phosphorylation. Present-day phosphate uptake by algae and bacteria seems to involve two transport systems: (a) An active transport process occurring at low external phosphate concentrations (as in unpollusive) process at higher phosphate concentrations (>10⁻⁶ M) (as in the interstitial water of reducing sediments). Laboratory model experiments are described for the reaction of reducing sugars with orthophosphate in the presence of cyanogen, producing glycosyl phosphates. These reactions proceed with appreciable yields only at high phosphate concentrations (>10⁻³ M), and may thus possibly serve as simulations of prebiological phosphorylation with diffusive transport, as it may have occurred in the interstitial water of reducing sediments.     

Selective inactivation of NADPH-cytochrome P-450 (c) reductase by diazotized 3-aminopyridine adenine dinucleotide phosphate

Cyanogen-bromide cleaved glucagon has been extensively purified in yields of 80–85% by the use of gel filtration and by cation-exchange chromatography at pH 4.5–5.2. This pH range maintains a charge difference between the holohormone and its cleavage product, the truncated homoserine lactone derivative, yet maintains the integrity of the lactone ring. Purity is determined by the lack of methionine and the presence of homoserine following peptide hydrolysis. The homoserine lactone is opened by treatment with 0.2 n triethylamine at pH 9.5. The lactone can be reformed by treatment with trifluoroacetic acid for 1 h at room temperature although protection against photooxidation of tryptophan-25 must be provided. The homoserine lactone form binds less well to glucagon receptors than does the homoserine form. Adenylate cyclase is activated by the lactone to an extent comparable to that obtained by native hormone but at elevated concentrations. The procedures described may be useful for purification of other cyanogen bromide cleavage products and is useful for semisynthetic methods based upon cyanogen bromide-cleaved derivatives of glucagon.     

Effect of urea on the partial reactions and crystallization pattern of sarcoplasmic reticulum adenosine triphosphatase

Steady-state ATPase activity, calcium binding, formation of phosphorylated enzyme intermediate with ATP in the presence of Ca2+, or with Pi in the absence of Ca2+, and association of ATPase molecules into bidimensional crystals, were studied using vesicular fragments of sarcoplasmic reticulum. The vesicles were exposed to increasing concentrations of urea in order to produce stepwise perturbations of protein structure and to test the effect of such perturbations on the partial reactions and crystallization pattern of sarcoplasmic reticulum ATPase. It was found that low concentrations of urea produce specific inhibition of Pi binding and enzyme phosphorylation with Pi (but not with ATP). Intermediate concentrations of urea reduce calcium binding affinity and cooperativity, while the ability of the enzyme to be phosphorylated with ATP and to form dimeric arrays is retained. These observations demonstrate that the sarcoplasmic reticulum ATPase is sensitive to physical perturbations producing specific and reversible changes in the Pi and calcium binding domains. These changes interfere with enzyme turnover, indicating that conformational effects related to binding and dissociation of Pi and calcium are tightly coupled to catalysis and energy transduction. Higher concentrations of urea produce irreversible denaturation, accompanied by total inhibition of calcium binding, enzyme phosphorylation with ATP, and association of ATPase chains in bidimensional crystals. Under these conditions, protein unfolding is manifested by a sharp reduction in the fluorescence of intrinsic tryptophan residues and of a covalently bound probe. These observations suggest that dimeric association and a tendency to form bidimensional crystals correspond to a basic property of the enzyme, which is linked to its native structure and whose character may change in the presence of ligands and/or during the catalytic cycle. On the other hand, the decavanadate-induced crystallization pattern cannot be interpreted in terms of a mechanistic relationship of ATPase dimerization with one of the intermediate states of the catalytic cycle.     

Thermal Synthesis of Nucleoside H-Phosphonates Under Mild Conditions

Nucleosides react rapidly with ammonium phosphite ((NH₄)₂HPO₃) at 60 °C to produce good yields of nucleoside-5-phosphite monoesters within 24 h. Under the same conditions, ammonium phosphate is unreactive, producing low yields of nucleotide only after extended reactions. These results confirm earlier suggestions that nucleoside H-phosphonates and their possible condensation products may have been produced on the primitive earth more easily than nucleotides.     

Amino acid sequence of beta-galactosidase. VII. Isolation of the 24 cyanogen bromide peptides.

All of the 24 cyanogen bromide peptides of beta-galactosidase have been isolated in pure form. Of these 8 ranged in size from 2 to 5 residues and were purified by paper electrophoresis. The 16 large peptides, from 23 to 119 residues, were chromatographed at pH 5.0 on a carboxymethyl-cellulose column in 0.02 M ammonium acetate buffer containing 8 M urea. A number of peptides were obtained in pure from following Sephadex G-50 or G-75 gel filtration. Others were separated on sulfopropyl-Sephadex or diethyl-(2-hydroxylpropylaminoethyl)-Sephadex. There large peptides were obtained in over 50% yield and several others were obtained in more than 25% yield.