Acid-labile amino acid precursors in the Murchison meteorite. 1: Chromatographic fractionation.

The amino acid content of a hot water extract of the Murchison meteorite can be increased by over 100 per cent by subjecting the extract to acid hydrolysis. The acid-labile compounds in the extract that account for this increase were fractionated by column chromatography on a cation exchange resin. Seventy mole per cent behaved as neutral or acidic compounds and were eluted from the column with an initial water wash. The remaining 30 mole per cent (basic precursors) were retained on the column and were eluted with the free amino acids by aqueous NH4OH. The acid-labile amino acid precursors in the water eluate could be retained and further fractionated on an anion exchange column, indicating that they are acidic compounds.     

Acid-labile amino acid precursors in the Murchison meteorite

The amino acid content of a hot water extract of the Murchison meteorite can be increased by over 100 per cent by subjecting the extract to acid hydrolysis. The acid-labile compounds in the extract that account for this increase were fractionated by column chromatography on a cation exchange resin. Sevently mole per cent behaved as neutral or acidic compounds and were eluted from the column with an initial water wash. The remaining 30 mole per cent (basic precursors) were retained on the column and were eluted with the free amino acids by aqueous NH₄OH. The acid-labile amino acid precursors in the water eluate could be retained and further fractionated on an anion exchange column, indicating that they are acidic compounds.Contribution number 101 from the Center for Meteorite Studies.     

Ethanol extraction of free amino acids from soil

Summary A technique was developed for extracting and analyzing the free amino acid fraction of soil. Ethanol was used as an extracting agent. Ethanolextraction curves showed 20 per cent ethanol was the optimum percentage for extraction. Extraction-time curves indicated 18 to 20 hours of extraction with 20 per cent ethanol produced satisfactory results.The free amino acid fraction of soil was characterized and the limitations of the technique were determined. The naturally occurring amino acids extracted with 20 per cent ethanol were limited to acidic and neutral amino acids; basic amino acids were not extracted in sufficient quantities to permit detection. Based on the percent recovery of amino acids incorporated into soil and extracted with 20 per cent ethanol 90 to 95 per cent of the acidic, 80 to 85 per cent of the neutral and 1 to 5 per cent of the basic amino acids used were recovered with the technique.     

The effect of nitrogen source on levels of amino acids in peas

Summary When nitrogen fixation in peas was partially replaced by nitrate assimilation as the source of nitrogen, an increase was found in the amount of soluble nitrogen that could be extracted from the fruits of the plants, and within this soluble fraction, increases were found in the levels of some of the acidic amino acids. Levels of protein amino acids in the peas were generally unaffected by the type of nitrogen source except for the level of aspartic acid which was about 20 per cent lower in peas supplied with nitrate. No differences were found in the proportions of the essential amino acids.     

Reactions between amino acid compounds and phenols during oxidation

Summary About 30 per cent of organic soil nitrogen can be hydrolized with HCl to amino acids; about 30 per cent is nonhydrolizable. In contrast to this high content of amino acid nitrogen is the small availability of the nitrogen to micro-organisms. In light of the theory proposing a reaction between the -amino group of amino acids or peptides and quinones formed during oxidation of lignin degradation products or other phenolic compound, different types of phenols were oxidized by phenolases in presence of amino acid compounds.It could be shown that the reaction of binding of nitrogen started at pH values higher than 6.5, and that only such phenols reacted which had no methoxylated hydroxyl groups. The reaction of some phenols during oxidation in presence of amino acids was accompanied by deamination and decarboxylation of the latter.The reaction products of phenols with amino acids were stable against hydrolysis. Using peptides it was found that all amino acids, except the N-terminal which is bound to oxidized phenols, could be hydrolyzed normally.With serum albumin it could be shown that there is a reaction with the amino group of the N-terminal amino acid and also with the -amino group of lysine residues with phenols during oxidation. The reacted protein seemed to be degraded normally with a protease ofBacillus subtilis.Guest Scientist as Fulbright Research Scholar from the Agronomy Department of the Iowa State University, Ames, Iowa, U.S.A.     

AMINO ACIDS IN SYNAPTIC VESICLES FROM MAMMALIAN CEREBRAL CORTEX: A REAPPRAISAL

Synaptic vesicles were prepared from rat cerebral cortex and separated by gel filtration from small molecular weight compounds contaminating this fraction. Electron microscopy of the vesicle suspension showed that vesicles were by far the most abundant morphological entities. The amino acid content of the purified vesicle fraction was examined and the two amino acids appearing in the most significant amounts were found to be taurine and glutamate. This amino acid pool was not osmotically sensitive as is the vesicular pool of ACh and remained attached to the vesicular protein after passage through Sephadex columns equilibrated in water. However, amino acids added to the vesicle fraction prior to passage through Sephadex did not become associated with this pool and this indicated that the vesicular pool was not likely to be an artifact due to the vesicular protein non-specifically adsorbing amino acids. The release of taurine from incubated synaptosome beds was studied and elevated medium K⁺ (56 mm ) was found to cause a small increase (36 per cent) in the amount of the taurine released to the medium. During the same experiments another physiologically active amino acid, glutamate, was released in more significant amounts, increasing in the medium by 186 per cent. The possible significance of the presence of taurine is discussed.     

The atmosphere of the primitive Earth and the prebiotic synthesis of amino acids

The atmosphere of the Earth at the time of its formation is now generally believed to have been reducing, an idea proposed by Oparin and extensively discussed by Urey. This atmosphere would have contained CH₄, N₂ with traces of NH₃, water and hydrogen. Only traces of NH₃ would have been present because of its solubility in water. UV light and electric discharges were the major sources of energy for amino acid synthesis, with electric discharges being the most efficient, although most other sources of energy also give amino acids.The first prebiotic electric discharge synthesis of amino acids showed that surprisingly high yields of amino acids were synthesized. Eleven amino acids were identified, four of which occur in proteins. Hydroxy acids, simple aliphatic acids and urea were also identified. These experiments have been repeated recently, and 33 amino acids were identified, ten of which occur in proteins, including all of the hydrophobic amino acids.Methionine can be synthesized by electric discharges if H₂S or CH₃SH is added to the reduced gases. The prebiotic synthesis of phenylalanine, tyrosine and trytophan involves pyrolysis reactions combined with plausible solution reactions.Eighteen amino acids have been identified in the Murchison meteorite, a type II carbonaceous chondrite, of which six occur in proteins. All of the amino acids found in the Murchison meteorite have been found among the electric discharge products. Furthermore, the ratios of amino acids in the meteorite show a close correspondence to the ratios from the electric discharge synthesis, indicating that the amino acids on the parent body of the carbonaceous chondrites were synthesized by electric discharges or by an analogous process.     

Properties of RNA fractions from nuclei of brain cells which stimulate incorporation of amino acids by brain ribosomes

Abstract— The properties of RNA fractions from nuclei of brain cells which were capable of stimulating amino acid incorporation into proteins of an homologous ribosomal system were investigated. RNA was routinely prepared from crude nuclear preparations of rat brain by a method which involved treatment with sodium dodecyl sulphate and phenol at 65°. The capacity of this preparation to stimulate incorporation of radioactivity from a mixture of 15 l -[¹⁴C]amino acids was greatly enhanced by preliminary incubation of the ribosomal system from brain for 5–20 min. The response was markedly dependent upon the concentrations of ribosomes and of the pH 5 fraction. The optimal level of Mg²⁺ for basal incorporation of amino acids into protein was 8 mm ; however, incorporation in the presence of nuclear RNA was greater at higher concentrations of Mg²⁺. The response to nuclear RNA was also enhanced as the K⁺ concentration was increased from 25 to 100 mm . The stimulatory effect of nuclear RNA on incorporation of l -[¹²C]eucine was either unaltered or depressed by addition of a mixture of 19 l -[¹²C]amino acids each at concentrations, of 10^?8, 10^?2, or 10^?1 mm . Under appropriate conditions of incubation, basal rates of incorporation and rates of incorporation stimulated by nuclear RNA were linear for 30 min. The response was proportional to the concentration of nuclear RNA between 34 and 136 μg. RNA prepared from ribosomes of rat brain essentially failed to stimulate incorporation of amino acids over this range of concentrations. Fractionation of nuclear RNA by centrifugation in sucrose density gradients revealed that 75 per cent of the stimulatory activity was in the fraction which sedimented below 12 S and contained about 25 per cent of the total RNA. Most of the remaining activity was in the 18 S region. Less than 5 per cent of the RNA in the lightest fraction (< 12 S) exhibited amino acid-acceptor activity, The stimulatory action of nuclear RNA on incorporation of amino acids was readily destroyed by mild treatment with pancreatic ribonuclease, whereas amino acid-acceptor activity was relatively resistant to this treatment. The results suggest that the brain may contain low molecular weight RNA with properties of messenger RNA.     

Extraction, purification and turnover of rat brain glycogen

Abstract— Glycogen was prepared from rapidly frozen rat brain by the usual techniques and found to contain considerable amounts of non-glycogen carbohydrate. The crude glycogen was partially purified by extraction with hot or cold water and reprecipitation. Enzymic estimation showed that the carbohydrate extracted into hot water contained only 50 per cent of glucose after hydrolysis; of the hot water insoluble material, namely some 30 per cent of the total carbohydrate present in the crude glycogen, less than half of the carbohydrate was released by hydrolysis in 1 M-HC1. The glycogen soluble in hot water incorporated ¹⁴C from [¹⁴C]glucose at considerably higher rates than the residual material and also decreased more rapidly during post-mortem autolysis. Glycogen extracted into cold water was of higher purity than that extracted by hot water; although the material behaved as glycogen during precipitation and re-extraction it contained only 75 per cent of its carbohydrate as glucose. Contaminants included fucose, galactose and hexuronic acid. The rates of metabolism of the partially purified glycogen are compared with published rates; it is suggested that the observed rates are inaccurate due to the impurities present in brain glycogen prepared by classical techniques.     

Some chemical properties of the HCl-methanol extract from the puparial cuticle of Drosophila melanogaster.

1. The HCl-methanol (HCl-MeOH) soluble fraction from the puparial cuticle of yellow, black and ebony of D. melanogaster was hydrolyzed in hydrochloric acid and examined for beta-alanine, ketocatechol, and acetic acid. 2. Between beta-alanine and ketocatechol and between beta-alanine and acetic acid, a quantitatively inverse relationship was found, respectively. The former relationship was further confirmed by the feeding experiment of beta-alanine to black. 3. Of total beta-alanine in the HCl-MeOH extract, the proportion of those having free amino group was 74.8 per cent. 4. All these results indicate that the HCl-MeOH soluble fraction of the puparial cuticle may be useful for investigating the cross-link structure of the cuticle.     

Characterization of methylated neutral amino acids from Escherichia coli ribosomes.

The methylated neutral amino acids from both 30S and 50S ribosomal subunits of an Escherichia coli K strain were characterized. The 50S ribosomal subunit contains three methylated neutral amino acids: N-monomethylalanine, N-monomethylmethionine, and an as yet unidentified methylated amino acid found in protein L11. Both N-monomethylalanine and N-monomethylmethionine were found in protein L33. The amount of N-monomethylmethionine in this protein, however, is variable but not more than 0.25 molecules per protein. Thus protein L33 from this E. coli K strain has heterogeneity in its N-terminal amino acid and can start with either N-monomethylalanine or N-monomethylmethionine. The N-monomethylmethionine residue was not derived from the reduction of N-formylmethionine in the protein. The 30S ribosomal subunit contains only one methylated neutral amino acid: N-monomethylalanine.     

Delivery of extraterrestrial amino acids to the primitive Earth. Exposure experiments in Earth orbit.

A large collection of micrometeorites has been recently extracted from Antarctic old blue ice. In the 50 to 100 micrometers size range, the carbonaceous micrometeorites represent 80% of the samples and contain 2% of carbon. They might have brought more carbon to the surface of the primitive Earth than that involved in the present surficial biomass. Amino acids such as "-amino isobutyric acid have been identified in these Antarctic micrometeorites. Enantiomeric excesses of L-amino acids have been detected in the Murchison meteorite. A large fraction of homochiral amino acids might have been delivered to the primitive Earth via meteorites and micrometeorites. Space technology in Earth orbit offers a unique opportunity to study the behaviour of amino acids required for the development of primitive life when they are exposed to space conditions, either free or associated with tiny mineral grains mimicking the micrometeorites. Our objectives are to demonstrate that porous mineral material protects amino acids in space from photolysis and racemization (the conversion of L-amino acids into a mixture of L- and D-molecules) and to test whether photosensitive amino acids derivatives can polymerize in mineral grains under space conditions. The results obtained in BIOPAN-1 and BIOPAN-2 exposure experiments on board unmanned satellite FOTON are presented.     

GLUCOSE AND AMINO ACID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELL FRACTIONS IN VITRO

Abstract—

• 1 The metabolism of three substrates, [U-¹⁴C]glucose, [U-¹⁴C]pyruvate and [U-¹⁴C]glutamate has been studied in vitro in neuronal and glial cell fractions obtained from rat cerebral cortex by a density gradient technique.
• 2 The mixed cell suspension, after washing, metabolized glucose and glutamate in a manner essentially similar to the tissue slice. Exceptions were a reduced ability to generate lactate from glucose and alanine from glutamate, and a lowered effect of added glucose in suppressing the production of aspartate from glutamate.
• 3 After 2 hr incubation with [U-¹⁴C]glucose, the concentration of the amino acids glutamate, glutamine, GABA, aspartate and alanine were raised in the neuronal, compared to the glial fraction to 234 per cent, 176 per cent, 202 per cent, 167 per cent and 230 per cent respectively although both were lower than in the tissue slice. Incorporation of radio-activity was absolutely lower in the neuronal fraction, however, and the specific activities of the amino acids were: glutamate 12 per cent, GABA 18 per cent, aspartate 34 per cent, and alanine 33 per cent of those in the glial fraction.
• 4 After the incubation with [U-¹⁴C]pyruvate, the pool size of the amino acids were higher than after incubation with glucose, except for GABA, which was reduced to one-third. The concentrations of the amino acids glutamate, glutamine, GABA, aspartate, and alanine in the neuronal fraction were respectively 46 per cent, 143 per cent, 105 per cent, 97 per cent, and 57 per cent of those in the glial. Thus, with the exception of alanine, the specific activity of the neuronal amino acids compared to the glial was little increased when pyruvate replaced glucose as substrate.
• 5 After 2 hr incubation with [U-¹⁴C]glutamate in the presence of non-radioactive glucose, the pool sizes of all the amino acids were increased in both neuronal and glial fractions, with the exception of neuronal alanine and glial glutamine. The concentrations of the amino acids glutamine, GABA, aspartate and alanine were raised in the neuronal fraction, compared to the glial, to 425 per cent, 187 per cent, 222 per cent, and 133 per cent respectively. The specific activities of all the amino acids were higher than with glucose alone with the exception of alanine, and neuronal GABA. Neuronal glutamine and aspartate had specific activities respectively 102 per cent and 84 per cent of glial.
• 6 An unidentified amino acid, with R_F comparable to that of alanine and specific activity close to that of glutamate, was also present after incubation. It was relatively concentrated in the neuronal fraction.
• 7 The distribution of the enzymes glutamate dehydrogenase, aspartate aminotransferase, glutamate decarboxylase and glutamine synthetase between the cell fractions was studied. With the exception of glutamine synthetase, none of the enzymes was lost from the cell fractions during their preparation. Only 14 per cent of the glutamine synthetase, compared with 75 per cent of total protein, was recovered in the fractions. Of the enzymes, glutamate dehydrogenase activity was 406 per cent, and glutamate synthetase activity 177 per cent in the neuronal fraction compared to the glial in the absence of detergent. In the presence of detergent, glutamate dehydrogenase control was 261 per cent, aspartate aminotransferase activity 237 per cent is the neuronal as compared to the glial fraction.
• 8 Incorporation of radioactivity into acid-insoluble material from either glutamate or pyruvate was twice as high into the neuronal as the glial fraction.
• 9 The extent to which these differences may be extrapolated back to the intact tissue is considered, and certain correction factors calculated. The significance of the observations for an understanding of the compartmentation of amino acid pools and metabolism in the brain, and the possible identification of such compartments, is discussed.

     

Protein and free amino acids in field-grown cowpea seeds as affected by water stress at various growth stages

Summary This study was undertaken to evaluate water stress effects during vegetative, flowering, and podfilling stages of cowpea plants (Vigna unguiculata L.) grown under natural field conditions in southern California on seed yield and protein and free amino acid content of the cowpea seeds. The lowest concentration of N was found in the seeds of the control treatment plants while the seed yield from these treatments was the highest as compared with the N concentration and yield of seeds from plants subjected to water stress during flowering and podfilling stages. The concentration of N in the seeds was inversely related to the seed dry weight yield. Protein arginine,-threonine,-serine,-cystine,-valine,-methionine, and-isoleucine were significantly affected by water stress at the three growth stages. There was no consistent pattern in the effect of water stress on the individual amino acids. The sum of protein amino acids in the cowpea seeds was not significantly influenced by the various treatments since some of the protein amino acids increased and others decreased producing an averaging effect on the figures comprising the sums of the amino acids. Water stress during the flowering and pod-filling stages increased the free amino acid pool, and at the same time, inhibited incorporation of the amino acids into the protein chain-thus lowering the protein amino acid fraction simultaneously. With the exception of methionine plus cystine, the essential amino acids in the seeds were present at concentrations equal to or greater than recommended by the World Health Organization and FAO. It is of particular importance to note that the concentration of lysine in the cowpeas was substantially higher than that found in wheat grain. It is also important to note that the amount of essential amino acids per gram of protein was not measurably affected by the water stress treatments during any of the growth stages.     

Studies on Roasting Changes of Proteins

Changes of casein and lysozyme during roasting at various times and temperatures 100 ~ 300°C), especially those in amino acid composition and formation of some nonvolatile degradation products, were investigated.

Tryptophan, methionine, basic amino acids and β-hydroxy amino acids were easily decomposed as compared with acidic amino acids and other neutral amino acids in casein and Iysozyme. In hot water extract of roasted casein were detected some free amino acids, peptides, organic acids such as α-ketoglutaric, tartaric and malic acids, and indole. It is considered that free amino acids are produced mainly through ionic cleavage of peptide bond with the water bound within casein.     

A precise method for the quantitation of proteins taking into account their amino acid composition.

A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-μl solutions containing 1 to 100 μg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per aliquot of hydrolysate analyzed can be determined.     

The effect of insulin on amino acid incorporation into exocrine pancreatic cells of the rat.

The rate of incorporation of radioactive leucine per cell in the acinar pancreatic cells of the rat increases by 50 per cent within one hour after subcutaneous administration of insulin, an effect that lasts for at least one more hour. The rate of incorporation has been measured by quantitative radioautography and by determination of the radioactivity per mug DNA in TCA-precipitable material from tissue homogenates. The capacity for amino acid (leucine and lysine) incorporation as measured by incubating pancreatic fragments in vitro is not enhanced by insulin treatment of the rat in vivo during one or more hours. Insulin was found to lower the serum concentration of most amino acids significantly, leucine by 50 per cent. The apparent effect of insulin on the incorporation of radioactive leucine in vivo can be explained by the difference in the specific radioactivity of the circulating amino acid in the treated rats as compared to the untreated ones. A change in amino acid concentration in the serum may likewise be the explanation of the decrease in amino acid incorporation rate in alloxan diabetic rats. The absence of a short term effect of insulin on the rate of protein synthesis does not exclude a long term effect as suggested by the higher rate of incorporation in the cells of peri-insular acini.     

抗植物病毒农药“病毒煞”的氨基酸成分分析

本文通过对抗植物病毒农药“病毒煞”的氨基酸成分进行分析,共含有１８种氨基酸,其中脯氨酸、谷氨酸、精氨酸和天冬氨酸含量较高,约占水解氨基酸总量的４７％;含有１９种游离氨基酸,脯氨酸含量最高,占游离氨基酸总量的５１％,说明氨基酸可能是其防病增产的有效成分之一。     

Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits.

Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.     

AMINO ACID INCORPORATION IN VITRO INTO PROTEIN OF NEURONAL AND GLIAL CELL-ENRICHED FRACTIONS

Slices of rabbit cerebral cortex were incubated in the presence of labelled amino acids. Following incubation, neuron- and gliaenriched fractions were obtained by density gradient centrifugation and the TCA-insoluble radioactivity determined. The protein-bound radioactivity was five to six times higher in the neuronal-enriched fraction than in the glial-enriched fraction after incubation with tritiated leucine. The neuronal fraction incorporated also a number of other amino acids to a higher extent than the glial fraction (neuron/glia ratio 2·5-6). A definite dependence of incorporation on the rate of oxygenation was demonstrated. The suppression of amino acid incorporation was more marked for the neuronal fraction than for the glial fraction during incubation in relative hypoxia. An increase of potassium concentration in the incubation medium enhanced the amino acid incorporation in both fractions. Low sodium levels decreased the incorporation. Puromycin inhibited incorporation to approximately 30 per cent of control for both fractions. Addition of cycloheximide and dinitrophenol resulted in greater inhibition of incorporation in the neuronal fraction than in the neuroglial fraction. Actinomycin D did not markedly affect the incorporation in any fraction. These results are discussed in relation to in vivo and in in vitro differences for transport and incorporation of amino acids.