Analytical Procedures for the Sequential Extraction of 14C-Labeled Constituents from Leaves, Bark and Wood of Cottonwood Plants

A procedure for the extraction, separation, and measurement of photosynthetically fixed ¹⁴C in up to 8 chemical fractions (CHCl), amino acids, organic acids, sugars, protein, starch, hemicellulose, and residue) from small samples (1 to 100 mg) of cottonwood (Populus deltoides Bartr.) leaf material is described. The different chemical fractions are extracted in a sequence of chemical, ion exchange, and enzymatic steps. The ¹⁴C-activity in these major fractions is then determined with liquid scintillation spectrometry. These major fractions (e.g., sugars, amino acids, organic acids) can be further separated into their individual chemical components by standard thin-layer or gas- chromatographic methods for quantitative analysis if specific activities are desired. The major advantage of the procedure is that many chemical fractions can be sequentially separated with good reproducibility from a small amount of plant material without transfer of the material from the original homogenizer or centrifuge tube.     

Effect of the prolonged administration of chlortetracycline on the content of free amino acids and low-molecular peptides in the blood serum of rats]

Five individual ninhydrin positive fractions were found in the blood serum of rats in the control group with the help of high voltage electrophoresis in combination with preparative high voltage electrophoresis and paper chromatography. An analogous analysis of the blood serum of the test animals treated with 50 mg/kg of chlortetracycline for 12 days revealed 1 new fraction in the weak basic region. After the use of chlortetracycline for 18 days 2 new fractions in the strong acid region, 1 new fraction at the level of the electrophoretic mobility of asparaginic acid and 2 fractions in the region between neutral and basic amino acids were found. Analysis of the above fractions showed that the blood sera of the control and test rats contained in addition to the free amino acids peptides consisting of 3 to 9 amino acid residues. Some peptides isolated from the sera of the test animals contained amino acids, such as ornithine and citrulline which are not usually contained in proteins.     

Organic Constituents and Complexation of Nickel(II), Iron(III), Cadmium(II), and plutonium(IV) in Soybean Xylem Exudates

The xylem exudates of soybean (Glycine max cv Williams), provided with fixed N, were characterized as to their organic constituents and in vivo and in vitro complexation of plutonium, iron, cadmium, and nickel. Ion exchange fractionation of whole exudates into their compound classes (organic acid, neutral, amino acid, and polyphosphate), followed by thinlayer electrophoresis, permitted evaluation of the types of ligands which stabilize each element. The polyvalent elements plutonium(IV) and iron(III) are found primarily as organic acid complexes, while the divalent elements nickel(II) and cadmium(II) are associated primarily with components of the amino acid/peptide fraction. For plutonium and cadmium, it was not possible to fully duplicate complexes formed in vivo by back reaction with whole exudates or individual class fractions, indicating the possible importance of plant induction processes, reaction kinetics, and/or the formation of mixed ligand complexes. The number and distribution of specific iron- and nickel-containing complexes varies with plant age and appears to be related to the relative concentration of organic acids and amino acids/peptides being produced and transported in the xylem as the plant matures.     

Studies on proteins of animal ribosomes

Summary Total ribosomal protein from rat liver ribosomes can be separated into about 20 chief electrophoretic fractions by preparative polyacrylamide gel electrophoresis. Ten electrophoretically homogeneous fractions have been isolated from the total mixture of ribosomal protein, respectively from proteins, prefractionated by CM-cellulose chromatography. Amino acid composition and molecular weights of some fractions have been determined. The amino acid composition of these fractions and of the total protein mixture are basically similar but there are also significant differences with regard to some amino acids. The molecular weights of the proteins studied are in the range between 7,000 and 29,000.     

Complete amino acid sequence of a subunit from rapeseed high molecular weight protein

A subunit (Mr 15,600) from the high molecular weight protein from rapeseed was separated and isolated; its purity and homogeneity were ascertained. The subunit was cleaved with cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease. The fragments were separated and isolated by polyacrylamide gel electrophoresis, gel filtration, column chromatography on Dowex 1 x 2, and paper electrophoresis. The amino acid compositions of the intact subunit and different fragments obtained from enzymatic and chemical cleavages were determined. The subunit and its fragments were sequenced by manual Edman method. The phenylthiohydantoin amino acids obtained after each step were identified by thin-layer chromatography and ultraviolet spectroscopy. The complete amino acid sequence of the subunit consisting of 125 amino acid residues has been established by the overlapping method.     

Methods for the identification of N-asparaginyl and O-seryl/threonyl glycosidic linkages to aminosugars in glycoproteins

Methods are presented for the identification of certain glycopeptide bonds in glycoproteins. Mucin-type linkages are determined following treatment of glycoproteins with alkaline sodium [³H]borohydride. Such treatment cleaves O-glycosidic bonds to serine and threonine and simultaneously labels the sugar and amino acid components of the linkage. Following acid hydrolysis and dansylation, the sugar component of the linkage is identified as its corresponding dansyl-hexosaminitol by fluorographic techniques. A method is described for the separation of dansyl-galactosaminitol and dansyl-glucosaminitol by thin-layer electrophoresis in borate buffers. The amino acid component of the glycopeptide linkage is identified by fluorography following two-dimensional thin-layer chromatography of its dansyl derivative on polyamide plates. For the analysis of plasma-type glycoproteins, glycopeptides are prepared by exhaustive pronase digestion and purified by gel filtration chromatography. Final purification is effected by dansylation and thin-layer electrophoresis. The linkage compound 2-acetamido-1-N-β-l-aspartyl-2-deoxy-β-d-glucopyranosylamine is isolated from such glycopeptides as its dansyl derivative following partial acid hydrolysis. Its identity is confirmed by comparison of its properties with those of the synthetic compound. Thus the components of the glycosylamine linkage are identified following complete acid hydrolysis, redansylation, and separation by thin-layer electrophoresis.     

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.     

Amino Acid composition of germinating cotton seeds

Total and free amino acid composition of germinating cotton seeds (Gossypium hirsutum L.) was determined. The germinating seeds were separated into cotyledon and developing axis fractions daily and the composition of each tissue was summed to get the whole seed composition. By separating the developing seeds into these two tissue fractions, and determining total and free amino acids, a balance sheet was developed for each amino acid. This technique allowed changes in distribution with time of each amino acid to be followed in each tissue. Data for total content and amount in protein of each amino acid are presented. Asparagine increased in the whole seed, and most of this increase was found in the free pool of the developing axis. Other amino acids (e.g. arginine, glutamic acid) increased in the free pool but showed an over-all decrease, indicating that they were being metabolized. Amino acid contents of storage and nonstorage protein isolates were determined.     

Determination of content and specific activity of amino acids in central nervous system tissue utilizing tritium and carbon-14 dual labeling

A method is reported for measuring content and specific activity of amino acids in central nervous system tissue of the rat. Tritiated dinitrofluorobenzene is used to produce dinitro-[³H]phenyl amino acid derivatives which are separated by two-dimensional thin-layer chromatography on silica gel. Both content and specific activity can be determined simultaneously by pulse-labeling with an appropriate ¹⁴C precursor and measuring radioactivity in the individual spots of dinitrophenyl amino acids scraped from the chromatogram. This method is sensitive to at least 100 pmol and is shown to be more rapid than previously described procedures.     

N5-(1-carboxyethyl)-ornithine, a new amino acid from the intracellular pool of Streptococcus lactis.

Intracellular concentrations of amino acids were determined in cells of Streptococcus lactis 133 during growth in complex, spent, and chemically defined media. Glutamic and aspartic acids represented the major constituents of the amino acid pool. However, organisms grown in spent medium or in defined medium supplemented with ornithine also contained unusually high levels of two additional amino acids. One of these amino acids was ornithine. The second compound exhibited properties of a neutral amino acid by coelution with valine from the amino acid analyzer. The compound did not, however, comigrate with valine or any other standard amino acid by two-dimensional thin-layer chromatography. The unknown amino acid was purified by paper and thin-layer chromatography, and its molecular structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy. This new amino acid was shown to be N5-(1-carboxyethyl)-ornithine. The 14C-labeled compound was formed by cells of S. lactis 133 during growth in spent medium or defined medium containing [14C]ornithine. Formation of the derivative by resting cells required ornithine and the presence of a metabolizable sugar. N5-(1-Carboxyethyl)-ornithine was synthesized chemically from both poly-S-ornithine and (2S)-N2-carbobenzyloxy-ornithine as a 1:1 mixture of two diastereomers. The physical and chemical properties of the amino acid purified from S. lactis 133 were identical to those of one of the synthetic diastereomers. The bis-N-trifluoroacetyl-di-n-butyl esters of the natural and synthetic compounds generated identical gas chromatography-mass spectrometry spectra. A mechanism is suggested for the in vivo synthesis of N5-(1-carboxyethyl)-ornithine, and the possible functions of this new amino acid are discussed.     

Structural similarities between the major polypeptides of thylakoid membranes from Chlamydomonas reinhardtii

The major polypeptides of thylakoid membranes from Chlamydomonas reinhardtii were purified by preparative gel electrophoresis and examined for structural similarities. The largest of these polypeptides has an apparent molecular mass of 29,500 ± 500 daltons, whereas the other two both have an apparent mass of 26,000 ± 500 daltons. The amino acid compositions and uv-absorption spectra of the 29K- and 26K-dalton polypeptides are very similar. The same pattern of release of amino acids was obtained from both fractions by digestion with carboxypeptidase Y. Endoproteolytic digestion with trypsin, chymotrypsin, staphylococcal protease, and mild acid yielded identical patterns of N-terminal amino acids from both the 29K- and 26K-dalton polypeptides. However, different patterns of peptides were found after electrophoresis of fragments generated by digestion with staphylococcal protease. Conditions of electrophoresis were defined that permitted separation of the 26K-dalton fraction into two components, designated as polypeptides 16 and 17 in the identification system of Chua and Bennoun (1975, Proc. Nat. Acad. Sci. USA72, 2175–2179). Amino acid compositions of these two polypeptides are nearly identical. Polypeptide 16 contained N-terminal isoleucine, but no free N-terminal amino group was detected in polypeptide 17. Electrophoretic analysis of staphylococcal protease digests of these two polypeptides revealed significant differences in the patterns of peptides. These data confirm that there are three distinct major polypeptides in these membranes, which are present at nearly equal amounts. However, the data also suggest that significant similarities in amino acid sequence exist between these polypeptides.     

Effect of transient neonatal hypothyroidism on the free aspartic acid, glutamic acid, and GABA content of different central auditory regions in the rat

The aspartic acid, glutamic acid, and gamma-aminobutyric acid (GABA) contents were determined in four central auditory system regions in rats with transient neonatal hypothyroidism compared with control ones: the ventral and dorsal parts of the cochlear nucleus, the central nucleus of the inferior colliculus, the auditory cortex, and in an extra-auditory structure, the substantia nigra pars reticulata. The animals were sacrificed at 50 days of age, brain tissue samples were taken out by microdissection, and the free amino acids were extracted. The amino acid content was assessed by double-isotope labelling following two-dimensional thin-layer chromatography separation. GABA content was significantly decreased in both cochlear nucleus regions and glutamic acid was elevated in the inferior colliculus. Neonatal hypothyroidism had no significant effect on the aspartic acid levels in the regions studied. The results suggest an effect of neonatal hypothyroidism on regional contents of free amino acids known as candidate neurotransmitters in the auditory system.     

TRYPTIC PEPTIDES FROM BOVINE WHITE MATTER PROTEOLIPIDS

Abstract— The amino acid composition of the fractions obtained after tryptic digestion of performic acid oxidized and non-oxidized white matter proteolipids was studied. The acid-soluble fraction from the tryptic digest represented between 25 and 30% of the starting material and was relatively enriched in hydrophilic amino acids and deficient in hydrophobic amino acids. The acid-soluble peptides were separated by high voltage paper electrophoresis, and the amino acid compositions of 16 peptides were determined; three additional peptides were obtained from the acid-soluble digest of the oxidized proteolipid. The sequence of 7 peptides including the N- and C-terminal peptides is reported. The results suggest that the protein is segregated into hydrophilic and hydrophobic regions and that small hydrophilic regions are separated by large hydrophobic areas.     

The use of fluorescamine as a detection reagent in protein microcharacterization

With recent advances in protein microchemistry, compatible methods for the preparation and quantitation of proteins and peptides are required. Fluorescamine, a reagent which reacts with primary amino groups has been used successfully to detect amino acids, peptides, and proteins in various micromethods. This article discusses these methods which include (1) amino acid analysis of protein and peptide hydrolysates with postcolumn fluorescamine derivatization; (2) purification and characterization of proteins and peptides by reversed-phase HPLC with postcolumn fluorescamine derivatization; (3) purification of peptides by two-dimensional chromatography and electrophoresis on thin-layer cellulose with fluorescamine staining; and (4) electroblotting of protein bands from SDS-PAGE to glass fiber filters and polyvinylidene difluoride (PVDF) membranes with fluorescamine staining. In addition, this article also compares a postcolumn fluorescamine detection system with a UV detection system in the applications of amino acid analysis and reversed-phase HPLC protein/peptide analysis.     

Preparation of Peptides Containing Any Desired Amino Acid: Methionyl Peptides of Bovine Rhodopsin

A general method is described which allows the identification and preparation of peptides containing any amino acid of interest. The method has been applied to isolation of the methionyl peptides from a peptic digest of oxidized bovine rhodopsin. The peptide digestion mixture is first partially separated by ion exchange column chromatography. Location of peptides containing the desired amino acid is performed by amino acid analysis of acid hydrolyzed column fractions by high voltage paper electrophoresis. Peptides are further purified and prepared by peptide mapping, elution, and amino acid analysis using inexpensive high capacity techniques. Peptide sequencing is performed by a manual dansyl-Edman method well adapted for rapidly processing large numbers of samples. The methods are particularly well suited for detection and preparation of peptides containing amino acids for which there is no specific detection method.     

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex Lh-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not tryptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortisone which are not identical to any of the known metabolites.     

Dégradation d'acides aminés à l'interface eau-sédiment réconstitué d'une lagune méditerranéenne (Golfe du Lion)

Degradation of Amino Acids at a Simulated Water-sediment Interface of a Mediterranean Lagoon Environment, Golfe du Lion The degradation of two ¹⁴C and ³H labelled amino acids at the simulated water-sediment interface of a Mediterranean lagoon was studied. The four day experiments included the respiratory activity measurement and the study of incorporation processes of the radioactivity in specific organic fractions of the ¹⁴C arginine. After active mineralization occurred during the first day, the radioactivity in the acid-soluble fraction decreased and polycondensed products were progressively incorporated. The radioactivity of the ³H lysine was included mainly in the acid soluble fraction. The nature of the substrate, and the reducing conditions in the environment affect the radioactivity distribution in the organic fractions. The identification of amino acids in the acid-soluble and base soluble fractions shows that the labelled arginine and lysine and other amino acids in the acid soluble and base soluble fractions shows that the labelled arginine and lysine released from the initial compounds are important quantitatively.     

Metabolism of [2-14C] acetate to amino acids and proteins in segments of bean seedling roots

Summary ¹⁴C from [2-¹⁴C] acetate was found to be incorporated into soluble and protein amino acids in substantial amounts by bean root apices. The ¹⁴C was spread through a wide range of amino acids in both these fractions. Glutamic acid was found to be heavily labelled with ¹⁴C in both soluble and protein amino acid fractions. The data are discussed in relation to present ideas on transport and utilization of amino acids in root systems.     

Isolation and characterization of the three fractions (DE-I, DE-II and DE-III) of rat-liver Z-protein and the complete primary structure of DE-II

Three fractions (DE-I, DE-II and DE-III) of Z-protein (fatty acid binding protein) have been isolated from rat liver cytosol by DEAE-cellulose chromatography and characterized. They had the same molecular weight of 14000 and essentially identical amino acid composition. However, compositions of endogenous fatty acids were found to differ strikingly from one another. Long-chain fatty acids detected in DE-II were palmitic, stearic, oleic, linoleic and arachidonic acids. In contrast to DE-II, DE-III contained mainly arachidonic acid. Molar ratios of endogenous long-chain fatty acids to both DE-II and DE-III were estimated to be around 1.0. Unlike the latter two fractions, DE-I was virtually lipid-free. Analyses of the three fractions by polyacrylamide gel electrophoresis, electrofocusing and DEAE-cellulose chromatography before and after delipidation suggested that the difference between DE-I and DE-II was in part due to fatty acids bound to DE-II. In contrast, DE-III appeared to be somewhat different from these forms in its protein structure, though tryptic peptide mappings of the three fractions did not reveal clear differences among them. Analysis of the primary structure was made on the most abundant fraction, DE-II, to investigate the relationship among the three fractions and to other proteins. The protein was a single chain consisting of 127 amino acid residues and had a mostly acetylated NH2 terminus and a free sulfhydryl group. The complete sequence of Z-protein showed striking homology to cellular retinoid binding proteins and peripheral nerve myelin P2 protein, which indicated the presence of a new family of cellular lipid-binding proteins diverged from a common ancestor. A possible intragenic duplication of Z-protein was also suggested.     

不吸水链霉菌葡萄糖异构酶的物化表征

葡萄糖异构酶是一种催化葡萄糖异构为果糖的酶。本文用紫外吸收光谱、红外光谱、氨基酸组分分析、聚丙烯酰胺凝胶梯度电泳、SDS-聚丙烯酰胺凝胶电泳、超薄 层聚丙烯酰胺凝胶等电聚焦电泳技术研究了不吸水链霉菌嗜热亚种M1033菌株产生的葡萄糖异构酶的一些物化性质。结果表明由本实验室制备的均一葡萄糖异构酶的A₂₈₀与A₂₆₀的比值是1.76。它是由一个亚单位组成的酶分子。最小分子量是49000。pI值是5.2。氨基酸组分与其它来源的葡萄糖异构酶的氨基酸组分相比较有一些差异,其中Glu,Gly,ALa和Leu的含量都此其它异构酶的高,而Met,Trp,Asp,Thr则比其它葡萄糖异构酶的低。