Are polyphosphates or phosphate esters prebiotic reagents?

It is widely held that there was a phosphate compound in prebiotic chemistry that played the role of adenosine triphosphate and that the first living organisms had ribose-phosphate in the backbone of their genetic material. However, there are no known efficient prebiotic synthesis of high-energy phosphates or phosphate esters. We review the occurrence of phosphates in Nature, the efficiency of the volcanic synthesis of P₄O₁₀, the efficiency of polyphosphate synthesis by heating phosphate minerals under geological conditions, and the use of high-energy organic compounds such as cyanamide or hydrogen cyanide. These are shown to be inefficient processes especially when the hydrolysis of the polyphosphates is taken into account. For example, if a whole atmosphere of methane or carbon monoxide were converted to cyanide which somehow synthesized polyphosphates quantitatively, the polyphosphate concentration in the ocean would still have been insignificant. We also attempted to find more efficient high-energy polymerizing agents by spark discharge syntheses, but without success. There may still be undiscovered robust prebiotic syntheses of polyphosphates, or mechanisms for concentrating them, but we conclude that phosphate esters may not have been constituents of the first genetic material. Phosphoanhydrides are also unlikely as prebiotic energy sources. Correspondence to: S.L. Miller     

罗非鱼对微型生态系统营养物水平的影响

本文总结了罗非鱼不同放养密度的微型生态系统中N、P浓度及P分布动态观测结果。在罗非鱼的影响下,微型生态系统中氨氮、颗粒磷和总磷浓度不同程度地高于对照组,而正磷酸盐浓度和沉积物磷的量显著地低于对照组。不同密度组某些指标的观测值虽有显著差异,但未见任何指标依罗非鱼放养密度而有规律地变动。微型生态系统中正磷酸盐浓度同浮游动、植物密度和初级生产力显著相关,氨氮浓度与浮游植物密度之间亦有显著的相关关系。然而,浮游植物密度与总磷浓度之间不存在营养级联假说所预见的下行影响,相反有前者决定于后者的上行影响的趋向。微型生态系统中P分布的变化可揭示罗非鱼促进系统中营养物循环,从而加速其富营养化的主要机制。     

A colorimetric assay method to measure acetyl-CoA synthetase activity: application to woodchuck model of hepatitis virus-induced hepatocellular carcinoma

A new spectrophotometric method for quantitation of acetyl-CoA synthetase (ACAS) activity is developed. It has been applied for ACAS assay in the liver tissues of a woodchuck model of hepatitis virus-induced hepatocellular carcinoma (HCC). The assay is based on the established pyrophosphate (PPi) detection system. ACAS activity is indexed by the amount of PPi, the product of ACAS reaction system of activated form of acetate (acetyl-CoA) with ACAS catalysis. PPi is determined quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and 2-mercaptoethanol. PPi reacts with molybdate reagent to produce phosphomolybdate and PPi-molybdate complexes. 2-mercaptoethanol is responsible for color formation which has the peak absorbance at 580 nm. This method was sensitive from 1 to 20 nmol of PPi in a 380-mul sample (1-cm cuvette). A ten-fold excess of Pi did not interfere with the determination of PPi. To study the major metabolic pathways of imaging tracer [1-(11)C]-acetate in tumors for detection of HCC by Positron Emission Tomography (PET), the activity of one of the key enzymes involved in acetate or [1-(11)C]-acetate metabolism, ACAS was assayed by this newly developed assay in the tissue samples of woodchuck HCCs. A significant increase of ACAS activity was observed in the liver tissues of woodchuck HCCs as compared with neighboring regions surrounding the tumors (P<0.05). The respective ACAS activities in the subcellular locations were also significantly higher in HCCs than in the surrounding tissues (P<0.05) (total soluble fraction: 876.61+/-34.64 vs. 361.62+/-49.97 mU/g tissue; cytoplasmic fraction: 1122.02+/-112.39 vs. 732.32+/-84.44 mU/g tissue; organelle content: 815.79+/-100.77 vs. 547.91+/-97.05 mU/ g tissue; sedimentable fragment: 251.92+/-51.56 vs. 90.94+/-18.98 mU/ g tissue). The finding suggests an increase in ACAS activity in the liver cancer of woodchuck models of HCC as compared to that in the normal woodchuck liver. The developed assay is rapid, simple and accurate and is suitable for the investigation of ACAS activity under physiologic and pathophysiologic conditions.     

Unidirectional transport of orthophosphate across the envelope of isolated cauliflower-bud amyloplasts

Isolated amyloplasts from cauliflower (Brassica oleracea L. var botrytis) buds are able to export orthophosphate unidirectionally into the incubation medium. This orthophosphate transport appears to be protein-mediated, as indicated by the following observations: (i) low temperature and the presence of inhibitors of protein-mediated transport reduced the rate of orthophosphate export, and (ii) the rate of orthophosphate export became saturated with rising internal substrate concentrations. Micromolar concentrations of 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid inhibited the rate of unidirectional orthophosphate export, thus indicating the involvement of the amyloplastic glucose-6-phosphate (Glc6P)translocator in the unidirectional export of orthophosphate. The effect of rising concentrations of orthophosphate upon the activity of ADP glucose pyrophosphorylase in desalted extracts was determined. Orthophosphate given in concentrations similar to those measured in the amyloplastic stroma under conditions of steady-state rates of Glc6P-dependent starch synthesis inhibited the activity of ADP-glucose pyrophosphorylase significantly. However, even under strong limiting substrate conditions the residual activity was sufficient to catalyze the flux of carbon into starch. The maximal rates of orthophosphate transport (in the counter-exchange mode) by isolated spinach (Spinacia oleracea L.) chloroplasts and by isolated cauliflower-bud amyloplasts were also determined. These rates were compared with the maximal rates of undirectional orthophosphate export by these plastids. From these measurements we can conclude that, compared with spinach chloroplasts, isolated amyloplasts of cauliflower exhibit a fivefold greater ratio of unidirectional orthophosphate transport to maximal rate of orthophosphate transport in the counter-exchange mode compared to spinach chloroplasts. The determined rate of maximal unidirectional orthophosphate export is sufficient to catalyze the release of additional inorganic phosphate liberated in the amyloplastic stroma during the process of Glc6P-dependent starch synthesis.     

The relationship between phosphate status and photosynthesis in leaves

Spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.) were grown in hydroponic culture with varying levels of orthophosphate (Pi). When leaves were fed with 20 mmol·l^–1 Pi at low CO₂ concentrations, a temporary increase of CO₂ uptake was observed in Pi-deficient leaves but not in those from plants grown at 1 mmol·l^–1 Pi. At high concentrations of CO₂ (at 21% or 2% O₂) the Pi-induced stimulation of CO₂ uptake was pronounced in the Pi-deficient leaves. The contents of phosphorylated metabolites in the leaves decreased as a result of Pi deficiency but were restored by Pi feeding. These results demonstrate that there is an appreciable capacity for rapid Pi uptake by leaf mesophyll cells and show that the effects of long-term phosphate deficiency on photosynthesis may be reversed (at least temporarily) within minutes by feeding with Pi.Abbreviation Pi orthophosphate     

Changes of adenine nucleotide and orthophosphate concentrations in buds of deciduous trees during spring reactivation

Summary Concentrations of AMP, ADP, ATP, and inorganic phosphate (Pi) were determined in buds of five deciduous tree species (Acer pseudoplatanus, Alnus glutinosa, Fagus sylvatica, Fraxinus excelsior, Quercus robur) during spring reactivation from February to the middle of May. In closed buds of diffuse-porous wood trees (Acer, Alnus, Fagus), the content of adenine nucleotides (AdN) increased temporarily between the middle of February and the middle of March. The main increase of AdN concentration appeared either when buds became swollen (Fraxinus, Fagus, Quercus), or at the time of bud-break (Acer, Alnus). Pi content in general decreased during the course of reactivation. It was almost zero in buds of Quercus at bud-break and afterwards, but in Fraxinus Pi concentration rose when bud-break took place. The extremely low AdN content in Quercus buds is contrasted by a steep increase in AdN content in Fraxinus following bud-break. The decrease of AdN content in emerging leaves of Quercus and Fagus could be related to the high age of these trees.Abbreviations AdN adenine nucleotide(s) - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - PEP phosphoenolpyruvate - Pi orthophosphate Supported by Deutsche Forschungsgemeinschaft and by Bundesministerium für Forschung und Technologie, Federal Republic of Germany.     

不同氮、磷浓度对铜绿微囊藻生长、光合及产毒的影响

对一株从野外分离得到的铜绿微囊藻产毒株进行分批培养,在不同的氮磷条件下研究其生长、光合荧光及毒素含量的变化。结果表明:正磷酸盐浓度不变时,铵氮浓度的改变对铜绿微囊藻的生长有明显影响。叶绿素a(Chl.a)含量在铵氮浓度为1.83-18.3mg/L时明显较大;微囊藻毒素(包括MC-LR和MC-RR)的含量在铵氮浓度为1.83mg/L时达到最大;当铵氮浓度为0-1.83mg/L时,随着铵氮浓度升高,可变荧光FV和MC的产量均增大,同时MC异构体的种类增多;铵氮浓度过大对M.aeruginosa的生长、生理和产毒均有抑制作用。在另一组实验中,即铵氮浓度不变而正磷酸盐浓度增大时,Chl.a含量呈总体下降的趋势,并且与FV/Fm呈显著正相关关系(P<0.01,r=0.97),MC(MC-LR和MC-RR)的含量在正磷酸盐浓度小于0.56mg/L时明显升高,MC-LR与FV/Fm呈显著正相关关系(P<0.01,r=0.967)。       

In-gel precipitation of enzymatically released phosphate

The phosphate precipitation reaction using ammonium molybdate and triethylamine under low pH has been applied to gel-based assays for detecting phosphate-releasing enzymes. The sensitivity of the assay is 10 pmol Pi/mm2 of 1.5-mm-thick gel. The assay is applicable to enzymes with a wide range of optimal pH, from acid (pH 4.5) to alkaline phosphatase (pH 9.7), and to enzymes that use acid-labile substrates such as apyrase and glutamine synthetase. Using a negative staining approach, maltose phosphorylase, a phosphate-consuming enzyme, can also be detected. The assay was used to detect glutamine synthetase isoforms, separated by nondenaturing polyacrylamide gel electrophoresis from crude maize extracts. For downstream applications such as staining gels for proteins, the gels with precipitate should be incubated in 10 mM dithiothreitol or beta-mercaptoethanol until the precipitate is dissolved and then thoroughly washed in water. In comparison to calcium phosphate precipitation or the phosphomolybdate-malachite green method, this method is more sensitive. It is a very simple, rapid, versatile, reproducible, and inexpensive method that could be a useful tool in enzymological studies.     

Orthophosphate influx and efflux rates of Chlorella fusca measured in a continuous turbidostat culture with 32P under various conditions

The main objective of the experiments with Chlorella fusca strain 211-8b was to measure, with adequate time resolution, the unidirectional influx rates of phosphate into non-phosphate-starved algae under different steady state conditions (light, temperature, 3-phosphoglycerate influence) or following the addition of several photosynthesis and phosphate transport inhibitors (phenylmercuric acetate, p-chloromercuribenzoate, arsenate). the algae were cultivated in a phosphate rich medium in a continuous turbidostat culture. The phosphate exchange experiments with carrier-free ³²PO ₄ ^3- were performed directly in the continuous culture. The sampling intervals after the tracer addition were 15 s.For a continuous steady state culture grown in the light (25° C) the unidirectional influx rate measured with ³²P is 260 times higher than the net uptake rate (=influx minus efflux rate) calculated from the mass balance using the data of this culture. In all experiments, except the control experiment with trichloroacetic acid killed cells, the specific activity of the intracellular inorganic orthophosphate compartment oscillates around a constant mean value which never reaches the specific activity of the nutrient medium within the duration of the short-term experiments (7.5 min). The inhibitors strongly affect the characteristics of the oscillations. The unidirectional influx rates are constant. Oscillating flushing rates with unlabelled phosphate from a storage compartment have been postulates to explain the oscillations. Oscillating rates from the individual cells are apparently synchronized by an unknown mechanism.