Further studies on the biosynthesis of gramicidin S and proteins in a cell-free system from Bacillus brevis

1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.     

On the enzyme system obtained from some mutants of Bacillus brevis deficient in gramicidin S formation

Twenty mutants of Bacillus brevis which were deficient in gramicidin S formation were isolated by N-methyl-N′-nitrosoguanidine treatment. In addition to three groups which have been previously classified, further two groups were established according to their characteristics of amino acid activating enzymes concerned with gramicidin S formation. The fourth group mutants had a phenylalanine activating enzyme, but they had an enzyme complex from which one specific enzyme among proline, valine and leucine activating enzymes was deleted. Some of them also the ability to form d-phenylalanyl-l-prolyl diketpiperazine (DKP) even though they had phenylalanine and proline activating enzymes. The fifth group mutants contained both a phenylalanine activating enzyme and a complex of prodine, valine, ornithine and leucine activating enzymes like as a wild strain, but did not synthesize gramicidin S, and also one of them could not form even DKP.Combination of enzymes from DKP (+) mutants of the fourth or fifth groups with the first group mutant which had an intact proline, valine, ornithine and leucine activating enzyme complex showed gramicidin S formation, but the combination of enzymes from DKP (−) mutants except a proline activating enzyme minus mutant with the first group mutant could not synthesize gramicidin S.     

The biosynthesis of gramicidin S in a cell-free system

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of ¹⁴C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [¹⁴C]-leucine, -proline and -phenylalanine over a period of 4hr. With [¹⁴C]leucine, incorporation into gramicidin S takes place in the range pH6–9 with maximum incorporation at pH7·0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [¹⁴C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.     

Transport of amino acids in Lactobacillus casei by proton-motive-force-dependent and non-proton-motive-force-dependent mechanisms.

Lactobacillus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed. Inhibition studies indicated that there were at least five amino acid carriers, for glutamine, asparagine, glutamate/aspartate, phenylalanine, or branched-chain amino acids. Transport activities had pH optima between 5.5 and 6.0, but all amino acid carriers showed significant activity even at pH 4.0. Leucine and phenylalanine transport decreased markedly when the pH was increased to 7.5. Inhibitors which decreased proton motive force (delta p) nearly eliminated leucine and phenylalanine uptake, and studies with de-energized cells and membrane vesicles showed that an artificial electrical potential (delta psi) of at least -100 mV was needed for rapid uptake. An artificial delta p was unable to drive glutamine, asparagine, or glutamate uptake, and transport of these amino acids was sensitive to a decline in intracellular pH. When intracellular pH was greater than 7.7, glutamine, asparagine, or glutamate was transported rapidly even though the proton motive force had been abolished by inhibitors.     

Purification and characterization of neutrophil chemotactic factors of Streptococcus sanguis

Two neutrophil chemotactic factors were isolated from the culture filtrates of Streptococcus sanguis ATCC 10556 and were chemically characterized as N-terminal blocked peptides of low molecular weight. One of the factors consisted of proline, valine, methionine, isoleucine and leucine and the other of methionine, isoleucine, leucine and phenylalanine. In both factors, methionine was detected as the sole N-terminal amino acid, but the amino group was blocked. The removal of N-terminal methionine yielded several N-terminal amino acids, suggesting that S. sanguis produced several N-terminal blocked methionyl peptides, all of which could be chemotactically active.     

Effect of cypermethrin on the free amino acids pool in an organophosphorus-insecticide-resistant and a susceptible strain of Tribolium castaneum

1. Proline was found to be the major component of CTC-12 (44%) and FSS II (45%) strain.2. The cypermethrin treatment resulted in an increase in most of the amino acids of sixth instar larvae and all amino acids of adult beetles of CTC 12 strain.3. In the susceptible strain (FSS II), however, the tyrosine, phenylalanine and arginine increased, whereas serine, proline, glycine, alanine, valine, isoleucine, leucine and lysine were decreased significantly in the sixth instar larvae.4. In the FSS II adult beetles, only aspartic acid increased, while other amino acids either decreased (threonine, proline, glycine, alanine, valine, methionine, isoleucine, tyrososine, lysine, arginine) or remained unaffected (serine, glutamic acid, leucine, phenylalanine, histidine).     

Four homologous domains in the primary structure of GrsB are related to domains in a superfamily of adenylate-forming enzymes

The entire nucleotide sequence of the Bacillus brevis grsB gene encoding the gramicidin S synthetase 2, which activates and condenses the four amino acids proline, valine, ornithine and leucine has been determined. The gene contains an open reading frame of 13,359 bp which encodes a protein of 4453 amino acids with a predicted Mr of 510,287. The gene is located within the gramicidin S biosynthetic operon, also containing the genes grsT and grsA, whose nucleotide sequences have been determined previously. Within the GrsB amino acid sequence four conserved and repeated domains of about 600 amino acids (45-50% identity) have been identified. The four domains are separated by non-homologous sequences of about 500 amino acids. The domains also share a high degree of similarity (20-70%) with eight peptide synthetases of bacterial and fungal origin as well as with conserved sequences of nine other adenylate-forming enzymes of diverse origin. On the basis of sequence homology and functional similarities, we infer that those enzymes share a common evolutionary origin and present a phylogenetic tree for this superfamily of domain-bearing enzymes.     

Quantitative analysis of free amino acids and carbohydrates in spring barley anthers at different stages of maturity

The content of the carbohydrates glucose, fructose and sucrose was determined in spring barley anthers at different stages of maturity. During maturation the sucrose content of the anthers increased markedly. The following 17 free amino acids were detected in anthers of different stages of maturity: aspartic acid, glutamic acid, serine, alanine, arginine, leucine, isoleucine, lysine, α-aminobutyric acid, glutamine, proline, tyrosine, phenylalanine, valine, threonine, cystine and glycine. Quantitative analysis was only carried out in amino acids present in higher concentrations in the analysed samples. These were: aspartic acid, glutamic acid, α-aminobutyric acid, proline, serine, valine and glutamine, and a mixture of amino acids (leucine, isoleucine, valine and phenylalanine). The total content of free amino acids increased with increasing maturity of the anthers. However, not all amino acids followed contributed to this increase, but only proline, glutamic acid, aspartic acid and glutamine. A small difference was found in the variety Gopal in which the aspartic acid content did not increase significantly, but the content of the mixture of amino acids and serine did. With the exception of green anthers of the variety Firlbecks Union, proline was present in the highest concentration in all samples analysed.     

Evidence for a signal sequence at the N terminus of the common precursor to adrenocorticothrophin and beta-lipotropin in mouse pituitary cells

The precursor to corticotropin and beta-endorphin was synthesized in a reticulocyte cell-free system under the direction of mRNA from mouse AtT-20 pituitary tumor cells in the presence of [3H]proline, [3H]phenylalanine, [3H]leucine, [3H]valine, [3H]isoleucine or [35S]methionine. Automatic Edman degradation of the radioactive cell-free product showed the following N-terminal sequence: Pro-1, Met-2, Leu-11, Leu-12, Leu-13, Leu-15, Leu-16, Leu-17, Ile-21 and Val-23. The corticotropin-endorphin precursor was also labeled in AtT-20 cells with [3H]valine, [3H]leucine, [3H]tryptophan, [3H]serine, [35S]methionine or [35S]cysteine. Automatic Edman degradation of the radioactive intact cell form gave the following N-terminal sequence: Trp-1, Cys-2, Leu-3, Ser-5, Ser-6, Val-7, Cys-8, Leu-11, Leu-17, Leu-18 and tentatively Met-27. The sequence of the intact cell form from AtT-20 cells matches the sequence of the cell-free form of bovine pituitary precursor beginning at Trp-27, as determined by recombinant DNA technology [Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C. Y., Cohen, S. N., and Numa, S. (1979) Nature (Lond.) 278, 423-427]. The sequence of the mouse pituitary mRNA-directed cell-free translation product also matches the bovine precursor beginning at Pro-2. The results suggest that both the mouse and bovine precursors possess a signal sequence of 26 amino acids which is cleaved in intact cells. CNBr cleavage of [35S]cysteine-labelled intact cell precursor gave rise to an N-terminal fragment of a size compatible with the presence of a methionyl residue at or near position 27.     

Purification and characterization of neutrophil chemotactic factors of Streptococcus sanguis

Two neutrophil chemotactic factors were isolated from the culture filtrates of Streptococcus sanguis ATCC 10556 and were chemically characterized as N-terminal blocked peptides of low molecular weight. One of the factors consisted of proline, valine, methionine, isoleucine and leucine and the other of methionine, isoleucine, leucine and phenylalanine. In both factors, methionine was detected as the sole N-terminal amino acid, but the amino group was blocked. The removal of N-terminal methionine yielded several N-terminal amino acids, suggesting that S. sanguis produced several N-terminal blocked methionyl peptides, all of which could be chemotactically active.     

Channeling of vanadium in two species of fungi.

Aspergillus flavus and Penicillium italicum were cultivated on Dox medium amended with different concentrations of a vanadium salt; vanadium pentoxide (V2O5). The fungal isolates were found to absorb considerable quantities of vanadium. Fungal contents of carbohydrates and proteins were decreased by the presence of vanadium in the growth environment. Vanadium was incorporated into several types of low and high molecular weight protein(s) and peptides. In case of P. italicum, lysine, tyrosine, glycine, methionine, valine, alanine, proline, glutamic acid and histidine were detected in the fungal cell free extract. While in case of A. flavus, the following amino acids were detected: cystine, methionine, leucine, phenylalanine, glycine, alanine, tyrosine, lysine, histidine, arginine and valine.     

The Inhibitory Effects of Various Amino Acids on the Growth of Barley Seedlings

The effect of fifteen amino acids, supplied singly, on the growthof isolated germinating barley embryos in the presence of nitratehas been studied. The L forms of lysine, arginine, tyrosine,proline, threonine, methionine, leucine, and valine at concentrationsof either 1 or 2 mM have been found to inhibit fresh-weightaccumulation. The inhibition by valine is relieved by furtheraddition of isoleucine and that of leucine by the addition ofboth isoleucine and valine. These interrelations have been interpretedas suggesting that leucine and valine can inhibit acetolactateand acetohydrorybutyrate synthesis. The inhibition of tyrosinecan be relieved by phenylalanine and that of lysine by ornithineor arginine. The possible reasons for these interrelationshipsare discussed.     

Subcellular localization and kinetic properties of arginase from the liver of Genypterus maculatus

1. From the liver of the teleost fish Genypterus maculatus, a partially purified preparation of arginase was obtained and characterized. 2. The Km value for arginine was found to be 9.1 mM at pH 7.5 and 11.5 mM at the optimum pH of 9.5. At both pH values, competitive inhibition was caused by ornithine and lysine, whereas proline, leucine, valine and isoleucine caused a non-competitive inhibitory effect. Branched chain amino acids were more inhibitory than proline. 3. The enzyme was found localized in the mitochondrial matrix of the liver of Genypterus maculatus. It is suggested that this localization would be of importance in the use of arginine as an energy source.     

Variation in the free amino acid content of skeletal muscle of Ophicephalus punctatus (Bloch)

The skeletal muscle of Ophicephalus punctatus contains nine essential free amino acids, arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, valine and lysine, and eight non-essential amino acids, alanine, aspartic acid, cystine, glutamic acid, glycine, tyrosine, proline and serine. Histidine and lysine dominated the free amino acids pool. Seasonal variation was detected in the levels of histidine, arginine, leucine, phenylalanine, glycine, cystine and serine with highest values occurring in April and again in November. Changes were also detected in the concentrations of certain amino acids as the fish grew in size. Levels of free amino acids did not significantly differ between sexes. Factors effecting variation are discussed.     

Qualitative Characterization of Some Peptides Inducing Morphogenesis in the Nematode- Trapping Fungus Arthrobotrys oligospora

Some peptides inducing capture organ formation in Arthrobotrys oligospora were characterized with regard to their amino acid contents. The N-terminal, C-terminal and total amino acids were determined as their dinitrophenyl-derivatives by thin layer chromatography on silica gel in two different solvents. The amino acid composition was further confirmed by gas-liquid chromatography. The peptides investigated had a high proportion of non-polar and aromatic residues. Thus, leucine, isoleucine, valine, proline, and tyrosine were present in all the peptides. In addition, phenyl-alanine, glycine, and alanine occurred in some preparations. Tyrosine, valine, and phenylalanine were found in N-terminal position, and leucine or isoleucine were C-terminals.     

Changes in plasma amino acid concentrations with increasing age in patients with propionic acidemia

The objective of the study is to analyze plasma amino acid concentrations in propionic acidemia (PA) for the purpose of elucidating possible correlations between propionyl-CoA carboxylase deficiency and distinct amino acid behavior. Plasma concentrations of 19 amino acids were measured in 240 random samples from 11 patients (6 families) with enzymatically and/or genetically proven propionic acidemia (sampling period, January 2001–December 2007). They were compared with reference values from the literature and correlated with age using the Pearson correlation coefficient test. Decreased plasma concentrations were observed for glutamine, histidine, threonine, valine, isoleucine, leucine, phenylalanine and arginine. Levels of glycine, alanine and aspartate were elevated, while values of serine, asparagine, ornithine and glutamate were normal. For lysine, proline and methionine a clear association was not possible. Significant correlations with age were observed for 13 amino acids (positive correlation: asparagine, glutamine, proline, alanine, histidine, threonine, methionine, arginine; negative correlation: leucine, phenylalanine, ornithine, glutamate and aspartate). This study gives new insight over long-term changes in plasma amino acid concentrations and may provide options for future therapies (e.g., substitution of anaplerotic substances) in PA patients.     

Different binding sites for entry and exit of amino acids in whole cells of Mycobacterium phlei.

On the basis of mutual inhibition of uptake with different amino acids in whole cells of Mycobacterium phlei, it was demonstrated that the binding site of proline was different from those of all other amino acids studied. Other groups of amino acids share a common binding site: lysine, histidine, and arginine; valine, leucine, and isoleucine; tryptophan, tyrosine, and phenylalanine; glutamic acid and aspartic acid. The exit and entry processes were studied for proline, glutamine, and glutamic acid. It was observed that in each case the entry and exit processes were mediated by different membrane sites.     

Compartmentalization of amino acids in surfactant aggregates

Cationic amino acids, arginine and lysine partition differentially from water into aqueous micellar sodium dodecanoate. Conversely, partitioning of serine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, valine, leucine, phenylalanine and isoleucine do not vary appreciably. Partitioning from neat hexane into dodecylammonium propionate trapped water in hexane is, however, dependent upon both electrostatic and hydrophobic interactions. These results imply that the interior of dedecylammonium propionate aggregates is negatively charged and is capable of hydrogen bonding in addition to providing a hydrophobic enviroment. The solubilities of amino acids in neat hexane substantiate the previously derived amino acid hydrophobicity scale. Relevance of partitioning in these systems to the postulated selective amino acid compartmentalization is discussed.     

Studies on Amino Acid Sequence Determination from N-Terminal of Peptide by Methylthiohydantoin

Methylisothiocyanate reacts with the amino group of amino acid in alkaline solution to give methylthiocarbamyl amino acid. This is converted to the methylthiohydantoin derivative (MTH-amino acid) by subsequent acidification.

Gas chromatographical identification of 20 amino acids were examined by making MTH-amino acids. Among them, ten amino acids (glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, serine and threonine) were successfully identified. Aspartic acid and glutamic acid could be identified as the methyl esters of the MTH-amino acids.     

Active transport of nonpolar amino acids in Chromatium vinosum

The photosynthetic purple sulfur bacterium, Chromatium vinosum, takes up the amino acids, L-phenylalanine and L-leucine, via two apparently different electrogenic, H+/amino acid symports. Na+ serves as an allosteric modulator for leucine transport, lowering the Km for leucine from 66 to 15 microM. C. vinosum cells also contain a system that transports both isoleucine and valine. The isoleucine/valine system has the attributes of a H+/amino acid symport at pH less than 7.5 but appears to function as a H+/Na+ (Li+)/amino acid symport at pH greater than or equal to 7.5. Na+ gradients produce an allosteric lowering of the Km values for both isoleucine and valine, from 14 to 7 microM and from 34 to 17 microM, respectively. C. vinosum also accumulates D-alanine in an energy-dependent reaction. The transport process appears to involve the electrogenic cotransport of D-alanine and Na+. The Km value for D-alanine was determined to be 9 microM. Unlike the previously characterized C. vinosum L-alanine/Na+ symport, Na+ gradients did not affect the Km for D-alanine transport. L-Alanine and glycine, but not alpha-aminoisobutyric acid, act as competitive inhibitors for D-alanine transport.