Low Contents of Carbon and Nitrogen in Highly Abundant Proteins: Evidence of Selection for the Economy of Atomic Composition

Proteins that assimilate particular elements were found to avoid using amino acids containing the element, which indicates that the metabolic constraints of amino acids may influence the evolution of proteins. We suspected that low contents of carbon, nitrogen, and sulfur may also be selected for economy in highly abundant proteins that consume large amounts of the resources of cells. By analyzing recently available proteomic data in Escherichia coli, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, we found that at least the carbon and nitrogen contents in amino acid side chains are negatively correlated with protein abundance. An amino acid with a high number of carbon atoms in its side chain generally requires relatively more energy for its synthesis. Thus, it may be selected against in highly abundant proteins either because of economy in building blocks or because of economy in energy. Previous studies showed that highly abundant proteins preferentially use cheap (in terms of energy) amino acids. We found that the carbon content is still negatively correlated with protein abundance after controlling for the energetic cost of the amino acids. However, the negative correlation between protein abundance and energetic cost disappeared after controlling for carbon content. Building blocks seem to be more restricted than energy. It seems that the amino acid sequences of highly abundant proteins have to compromise between optimization for their biological functions and reducing the consumption of limiting resources. By contrast, the amino acid sequences of weakly expressed proteins are more likely to be optimized for their biological functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of different methods of acid hydrolysis on the nitrogen distribution in two soils

Summary An inorganic (Bainsville) and an organic (Farnham) soil were hydrolyzed by continuous and stepwise hydrolysis with hot 3N HCl for 1, 2, 3, 4, 11, 15, 18 and 24 h and continuously with hot 6N HCl for 24 h. The following nitrogen forms were determined: total N, hydrolyzable-N, amino acid-N, amino sugar-N and ammonia-N. Proportions of ‘unknown’ N were computed from these data. Continuous hydrolysis with 3N HCl yielded more amino acid-N and less ‘unknown’ N than did stepwise hydrolysis with the same acid strength. But continuous hydrolysis for 24 h with 6N HCl produced more amino acid-N and less ‘unknown’ N than did hydrolysis with 3N HCl by either method. It was estimated that 33 and 54% of the total N in the inorganic and organic soil, respectively, was protein-N. The ‘unknown’ N in the inorganic and organic soil constituted 51 and 37% of the total N, respectively. From our work it appears that the ‘unknown’ N is not proteinaceous. It can be readily degraded chemically and microbiologically to NH₃ and N-gases. More attention needs to be given to identifying the ‘unknown’ N which constitutes a large portion of the total soil-N. A more adequate knowledge of the chemical constitution of the ‘unknown’ soil N may lead to the development of technologies that will make more efficient use of the N in soils.     

尕海湿地退化演替过程中土壤有机氮组分的变化特征

为探究尕海湿地退化演替过程中土壤有机氮各组分变化规律,采用野外采样与室内分析相结合的方法,研究尕海湿地未退化(UD)、轻度退化(LD)、中度退化(MD)和重度退化(HD)4个退化演替阶段的土壤总氮(TN)和有机氮组分[未知态氮(HUN)、酸解氨态氮(AMN)、酸解氨基酸态氮(AAN)以及氨基糖态氮(ASN)]含量及其分布特征。结果表明: 当尕海湿地退化演替到LD时,0～10 cm层土壤TN、HUN、AMN和AAN含量分别降低17.3%、19.4%、8.6%和-5.6%,MD时分别降低28.0%、19.4%和17.1%和0,HD时分别降低35.8%、28.8%、28.6%和55.6%;10～20 cm层,LD时上述氮素含量分别降低4.0%、10.3%、2.9%和9.1%;MD时分别降低21.0%、18.3%、-2.9%和-9.1%;HD时分别降低9.9%、38.9%、21.2%和51.4%;而20～40 cm无显著变化;4个退化阶段各酸解氮组分占TN比例大小顺序为HUN(25.9%～32.5%)> AMN(6.7%～11.1%)> AAN(4.8%～11.1%)> ASN(1.2%～4.4%)。冗余分析显示,土壤含水量是土壤有机氮组分变化的主要驱动因子。尕海湿地退化显著降低了0～10 cm层土壤TN及酸解氮各组分含量,减弱了土壤氮“汇”功能,AAN和ASN对湿地退化最为敏感。     

半湿润农田生态系统长期施肥对土壤团聚体中有机氮组分的影响

通过对黄土高原半湿润农田生态系统25年的田间肥料定位试验,研究了长期不同施肥模式对土壤有机氮组分及其在各级团聚体中分布的影响.结果表明:长期施肥对水解氨态氮、水解未知氮在土壤各级团聚体中分布的影响最大,对氨基酸态氮的分布有一定影响,而对氨基糖态氮分布的影响较小.长期施用化肥和有机肥能有效地影响水解氨态氮和水解未知氮与团聚体的结合作用,而氨基糖态氮在土壤氮循环转化过程中具有较强的稳定性.长期施肥条件下土壤水解全氮与有机碳、全氮以及团聚体分形维数均呈极显著正相关,其r分别为0.942,0.981,0.910(P<0.001),说明土壤有机氮组分对土壤团聚体的形成和性质具有显著影响.相关分析表明,土壤全氮或有机质对1～2mm和0.25～1mm土壤团聚体中各有机氮组分的影响较大.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

METHYLATED AMINO ACID RESIDUES OF PROTEINS OF BRAIN AND OTHER ORGANS

—Methods for the determination of methyl-lysine, methyllarginine and methylhistidine residues of tissue proteins are described. They consist of preliminary purification of basic amino acids, enzymic removal of lysine, arginine and histidine followed by amino acid analysis. Recovery rates and specificities of the method were satisfactory. The contents of methylamino acids in proteins of mammalian organs were determined. The distribution of proteins containing the methylamino acids in human brain showed that the concentrations of methyl-lysine and N^G,N′^G-dimethylarginine were highest in the gray matter of the cerebellar cortex and relatively high in regions rich in gray matter, while those of N^G-mono- and N^G,N′^G-dimethylarginine were highest in the white matter. The following findings suggest that most of the N^G-mono- and N^G,N′^G-dimethylarginine was associated with the myelin basic protein. The distribution of the methylarginine residues of acid-soluble proteins in bovine brains coincided with the cerebroside pattern. The concentrations of the amino acids in acid-soluble proteins of rat brain increased concomitantly with the increase of cerebroside. The methylamino acid content in proteins increased during the purification of the myelin basic protein from the white matter of human and bovine brains. Proteins containing N^G,N^G-dimethyiarginine and di- and trimethyl-lysine are concentrated in cell nuclei. The first amino acid was found mainly in nucleoplasmic proteins and the other two were found in histones. The concentration of 3-methylhistidine residue, highest in muscular proteins, is low in cerebral proteins and is probably derived from proteins of walls of blood vessels in the brain.     

氨基酸添加对亚热带森林红壤氮素转化的影响

为探究氨基酸氮形态对亚热带土壤氮素含量及转化的影响,选择建瓯市万木林保护区的山地红壤为对象,采用室内培养实验法,通过设计60%和90%WHC两种土壤含水量并添加不同性质氨基酸,测定了土壤中铵态氮、硝态氮、可溶性有机氮的含量和氧化亚氮的释放量,分析了可溶性有机碳、土壤p H值的大小变化及其与氮素的相互关系。结果表明:与对照处理相比,氨基酸添加显著增加了土壤NH_4~+-N含量并使土壤p H值升高,且在一定程度上解除了高含水量(90%WHC)对NH_4~+-N产生的抑制,其中甲硫氨基酸的效果最为明显。酸性、碱性、中性氨基酸对土壤NO_3~--N含量和N_2O释放影响不显著,但甲硫氨基酸可显著抑制土壤硝化从而导致NH_4~+-N的积累,并在培养前期抑制土壤N_2O产生而在培养后期促进N_2O释放,总体上促进N_2O释放。60%WHC的氨基酸添加处理较90%WHC条件下降低土壤可溶性有机氮的幅度更大。氨基酸对土壤氮素转化的影响与带电性关系较小,而可能与其分解产物密切相关。可见,不同性质氨基酸处理对森林土壤氮素含量及转化存在不同程度的影响,且甲硫氨基酸对土壤氮素转化的影响机理值得深入研究。     

Comparative characterization of proteolytic enzymatic preparations by the extent of hydrolysis of microbial protein

Proteolytic enzymatic preparations obtained from fungi and bacteria were compared by their ability to hydrolyze yeast protein. Fungal preparations were more effective. There was a more than twofold increase in the level of amine nitrogen in cell biomass hydrolysates in comparison to that induced by bacterial preparations. The amino acid composition of these hydrolysates was studied. Amyloprotooryzin, a preparation fromAspergillus oryzae 387, displayed the highest potency in profound protein hydrolysis: the concentration of free amino acids released was 34.7% in comparison to 20.6% induced by amyloryzin and 10.5% by protosubtilin.     

How Prochlorococcus bacteria use nitrogen sparingly in their proteins

Organisms use proteins to perform an enormous range of functions that are essential for life. Proteins are usually composed of 20 different kinds of amino acids that each contain between one and four nitrogen atoms. In aggregate, the nitrogen atoms that are bound in proteins typically account for a substantial fraction of the nitrogen in a cell. Many organisms obtain the nitrogen that they use to make proteins from the environment, where its availability can vary greatly. These observations prompt the question: can environmental nitrogen scarcity lead to adaptive evolution in the nitrogen content of proteins? In this issue, Gilbert & Fagan (2011) address this question in the marine cyanobacteria Prochlorococcus, examining a variety of ways in which cells might be thrifty with nitrogen when making proteins. They show that different Prochlorococcus strains vary substantially in the average nitrogen content of their encoded proteins and relate this variation to nitrogen availability in different marine habitats and to genomic base composition (GC content). They also consider biases in the nitrogen content of different kinds of proteins. In most Prochlorococcus strains, a group of proteins that are commonly induced during nitrogen stress are poor in nitrogen relative to other proteins, probably reflecting selection for reduced nitrogen content. In contrast, ribosomal proteins are nitrogen rich relative to other Prochlorococcus proteins, and tend to be down‐regulated during nitrogen limitation. This suggests the possibility that decaying ribosomal proteins act as a source of nitrogen‐rich amino acids during periods of nitrogen stress. This work contributes to our understanding of how nutrient limitation might lead to adaptive variation in the composition of proteins and signals that marine microbes hold great promise for testing hypotheses about protein elemental costs in the future.     

Organic matter transformations in soils

Summary Post-incubation fractionation of soils incubated with C₁₄-tagged oat roots under aerobic and anaerobic conditions and chromatographic separation of hydrolysates of different organic matter fractions indicated the incorporation of C¹⁴-labelled amino acids in soil organic matter. Anaerobic incubation leads to the formation of hydrolysable heavily C¹⁴-labelled organic substances in greater quantity. The amino acid composition of the different fractions revealed not a very significant qualitative difference. The significance and causes of stabilization of amino acids in soil organic matter are discussed.     

长期施肥对红壤性水稻土有机氮组分的影响

通过16年的田间定位试验,研究了长期不同施肥模式对红壤性水稻土有机氮组分的影响.结果表明：长期化肥处理对土壤各氮素含量的作用不明显;有机物料循环特别是有机肥和化肥配施显著提高了土壤矿质氮和有机氮含量,提高幅度分别为55.2%和38.8%.有机物料循环处理显著提高了酸解性氮组分,其对土壤铵态氮、氨基糖氮和未知氮含量的提高幅度分别为36.5%、68.4%和73.9%;有机物料与化肥配施后,氨基酸氮含量也显著提高,提高幅度达71.1%,但是降低了未知氮含量,降低幅度为34.5%.此外,各施肥处理土壤累积矿化氮量均随培养时间的延长呈增加趋势,有机物循环或配施化肥处理土壤矿化氮量均高于单施化肥处理.     

Effect of N- and C-terminal deletions on the RNA N-glycosidase activity and the antigenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica

Karasurin-A, from root tubers of Trichosanthes kirilowii var. japonica, is a type I ribosome-inactivating protein (RIP) that displays activity of RNA N-glycosidase to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in the ribosome. We expressed recombinant proteins of karasurin-A and its various mutants with N- or C-terminal deletions in Escherichia coli as fusion proteins with maltose-binding protein (MBP), and compared their enzymatic activities and antigenicities. Muteins of karasurin-A generated by deleting either the first 100 N-terminal or the last 30 C-terminal amino acid residues lost activity of RNA N-glycosidase. The mutant proteins whose 80 N-terminal or 20 C-terminal amino acids were deleted could depurinate rRNA although the activities were decreased drastically. The antigenicities of the recombinant proteins were considerably reduced by deleting 20 amino acid residues from either N- or C-terminal regions.Revisions requested 30 September 2004; Revisions received 22 October 2004     

Amino acid contents and transport in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under different nitrogen conditions

Oilseed rape (Brassica napus L.) needs very high nitrogen fertilizer inputs. Significant amounts of this nitrogen are lost during early leaf shedding and are a source of environmental and economic concern. The objective of this study was to investigate whether the remobilization of leaf amino acids could be limiting for nitrogen use efficiency. Therefore, amino acid concentrations were analyzed in subcellular compartments of leaf mesophyll cells of plants grown under low (0.5 mM NO₃^–) and high (4 mM NO₃^–) nitrogen supply. With high nitrogen supply, young leaves showed an elevated amino acid content, mainly in vacuoles. In old leaves, however, subcellular concentrations were similar under high and low nitrogen conditions, showing that the excess nitrogen had been exported during leaf development. The phloem sap contained up to 650 mM amino acids, more than four times as much than the cytosol of mesophyll cells, indicating a very efficient phloem-loading process. Three amino acid permeases, BnAAP1, BnAAP2, and BnAAP6, were identified and characterized. BnAAP1 and BnAAP6 mediated uptake of neutral and acidic amino acids into Xenopus laevis oocytes at the actual apoplastic substrate concentrations. All three transporters were expressed in leaves and the expression was still detectable during leaf senescence, with BnAAP1 and BnAAP2 mRNA levels increasing from mature to old leaves. We conclude that phloem loading of amino acids is not limiting for nitrogen remobilization from senescing leaves in oilseed rape.     

Further evidence for the importance of residual leaf protein complexes in litter decomposition and the supply of nitrogen for plant growth

Summary The nitrogen absorbed by birch seedlings grown in sand culture has been used as a measure of the resistance to decomposition of complexes formed in vitro by interaction between protein and water-extractable leaf constituents ofCalluna vulgaris, Chamaenerion angustifolium andCircaea lutetiana. The resistance to decomposition of these complexes measured in this way isCalluna vulgaris >Chamaenerion angustifolium >Circaea lutetiana. This result seems to be in agreement with the soil-forming and field characteristics of these species and especially the raw humus-forming tendencies ofCalluna vulgaris.The nitrogen released, as measured by the nitrogen absorbed by birch seedlings, fromCalluna raw humus (H-layer material) and the model protein —Calluna leaf extractives complex is very nearly proportional to the widely different amounts of total nitrogen initially added to the root environment thereby suggesting that the organic nitrogen of the model complex and of theCalluna raw humus have similar properties.The amount of nitrogen absorbed by the birch seedlings when unaltered protein was added to the sand culture was less than when protein —Circaea lutetiana leaf extractives complex was added. This is ascribed to too rapid release and loss of nitrogen before it could be absorbed by the birch seedling roots and suggests a role for some leaf protein complexes in the conservation of litter nitrogen. Some possible effects of protein complexes in relation to the digestion of fresh and dry leaf material of various plant species by animals are discussed.Information from archeological observations and from investigations into the tanning of proteins is considered in relation to possible factors influencing the decomposition of protein complexes of litter and it seems probable that a less acid reaction,e.g. such as is brought about by the addition of adequate amounts of calcareous material, will assist the mobilization of the nitrogen of raw-humus-forming litter.The contention that the protein-precipitating substances of leaves are of fundamental importance in soil processes, especially raw humus formation and the supply of nutrients for plant growth, is considered to be supported by experimental approaches from different directions.     

Variations in the amino acid composition of cyanophycin in the cyanobacterium Synechocystis sp. PCC 6308 as a function of growth conditions

Gas chromatography-mass spectrometry studies of the nitrogen isotopic composition of the N-trifluoroacetyl n-butyl ester derivatives of the amino acids from isolated hydrolyzed cyanophycin from ¹⁵N-enriched cells led to two major findings: (1) the amino acid composition of this granular polypeptide, isolated using procedures optimized for extracting and purifying cyanophycin from cells in the stationary growth phase, varied with the culture growth condition; (2) the rate of incorporation of exogenous nitrate differed for each nitrogen atom of the amino acid constituents of cyanophycin or cyanophycin-like polypeptide. Arginine and aspartic acid were the principle components of cyanophycin isolated from exponentially growing cells and from light-limited stationary phase cells, with glutamic acid as an additional minor component. The cyanophycin-like polypeptide from nitrogen-limited cells contained only aspartic and glutamic acids, but no arginine. The glutamic acid content decreased and arginine content increased as nitrate was provided to nitrogen-limited cells. These cells rapidly incorporated nitrate at different rates at each cyanophycin nitrogen site: guanidino nitrogens of arginine>aspartic acid >-amino nitrogen of arginine>glutamic acid. Little media-derived nitrogen was incorporated into cyanophycin of exponentially growing cells during one cellular doubling time.Abbreviations asp-TAB, glu-TAB, arg-TAB N-Trifluoroacetyl n-butyl ester derivatives of aspartic acid, glutamic acid and arginine, respectively - CAP chloramphenicol - CF correction factor - TFAA Trifluoroacetic anhydride - MBTFA N-Methyl-bis-trifluoroacetamide     

Amino acid content of leguminous proteins as affected by genetic and nutritional factors. I.

Summary Tetraploid and diploid red clover have been cultivated in greenhouse under aseptic conditions. The plant cultures were given only one source of nitrogen: either they were given nitrate in the nutrient solution or they were inoculated withRhizobium for establishing symbiotic fixation.The amino acid composition of protein from the different series and from different parts of the plants was analysed by paper chromatography.There was a clear arginine reaction from the tetraploid clover material but no arginine at all was found in the diploid clover material.Plants fed on nitrate and those fed on symbiotically fixed nitrogen showed the same amino acid pattern as far as can be judged from the chromatograms. The hydrolysates from inoculated plants showed usually more intense ninhydrine reaction on the chromatograms than hydrolysates from not inoculated plants, although diluted to the same nitrogen content. For this fact we have so far no explanation.     

Vegetation cover regulates the quantity,quality and temporal dynamics of dissolved organic carbon and nitrogen in Antarctic soils

Populations of the two native Antarctic vascular plant species (Deschampsia antarctica and Colobanthus quitensis) have expanded rapidly in recent decades, yet little is known about the effects of these expansions on soil nutrient cycling. We measured the concentrations of dissolved organic carbon (DOC) and nitrogen (DON), amino acids and inorganic N in soils under these two vascular plant species, and under mosses and lichens, over a growing season at Signy Island in the maritime Antarctic. We recorded higher concentrations of nitrate, total dissolved nitrogen, DOC, DON and free amino acids in soil under D. antarctica and C. quitensis than in lichen or moss dominated soils. Each vegetation cover gave a unique profile of individual free amino acids in soil solution. Significant interactions between soil type and time were found for free amino acid concentrations and C/N ratios, indicating that vascular plants significantly change the temporal dynamics of N mineralization and immobilization. We conclude that D. antarctica and C. quitensis exert a significant influence over C and N cycling in the maritime Antarctic, and that their recent population expansion will have led to significant changes in the amount, type and rate of organic C and N cycling in soil.     

Intraspecific larval competition in two solitary parasitoids, Apoanagyrus (Epidinocarsis) lopezi and Leptomastix dactylopii

Analysis of total aromatic amino acid (free and bound) in some cucumber accessions selected previously for resistance to western flower thrips, Frankliniella occidentalis (Pergande) [Thysanoptera: Thripidae], indicated that low concentrations of these essential nutrients, relative to total leaf protein, were correlated with a reduction in damage by the insect. Further analysis of samples of four important horticultural crops (lettuce, tomato, pepper and cucumber) with unknown levels of resistance to thrips showed a significant genotypic variation in the concentrations of total aromatic amino acids relative to the total leaf protein. Accessions from each crop with low or high concentrations of aromatic amino acids in proteins were exposed to thrips larvae. Regression analysis showed a highly significant positive correlation between aromatic amino acid concentration in leaf protein and thrips damage, regardless of crop species. It is concluded that higher concentrations of aromatic amino acids in plant proteins are important for successful thrips development. These results provide plant breeders with a promising tool for indirect selection without using undesirable insect bioassays.     

Strategy for comprehensive identification of human N‐myristoylated proteins using an insect cell‐free protein synthesis system

To establish a strategy for the comprehensive identification of human N‐myristoylated proteins, the susceptibility of human cDNA clones to protein N‐myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an insect cell‐free protein synthesis system. One‐hundred‐and‐forty‐one cDNA clones with N‐terminal Met‐Gly motifs were selected as potential candidates from ～2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N‐myristoylation was evaluated using fusion proteins, in which the N‐terminal ten amino acid residues were fused to an epitope‐tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N‐myristoylated. The metabolic labeling experiments both in an insect cell‐free protein synthesis system and in the transfected COS‐1 cells using full‐length cDNA revealed that 27 out of 29 proteins were in fact N‐myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N‐myristoylated proteins that have not been reported previously to be N‐myristoylated, indicating that this strategy is useful for the comprehensive identification of human N‐myristoylated proteins from human cDNA resources.