The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo. Processing of altered iso-1-cytochromes c created by oligonucleotide transformation

The specificities of methionine aminopeptidase and amino-terminal acetylation in the yeast Saccharomyces cerevisiae were investigated in vivo by sequencing a series of altered iso-1-cytochrome c. Twenty iso-1-cytochromes c, each having a different penultimate residue in the sequence Met-Xaa-Phe-Leu-, were created by transforming yeast directly with synthetic oligonucleotides. The degree of methionine cleavage and amino-terminal acetylation was estimated from the levels of pertinent peptides separated by high performance liquid chromatography. The results confirmed our earlier hypothesis (Sherman, F., Stewart, J. W., and Tsunasawa, S. (1985) BioEssays 3, 27-31) that methionine is completely removed from penultimate residues having radii of gyration of 1.29 A or less (glycine, alanine, serine, cysteine, threonine, proline, and valine). However, only partial cleavage occurred in the sequences Met-Thr-Pro-Leu- and Met-Val-Pro-Leu-, demonstrating that proline at the third position inhibits methionine cleavage when the penultimate residue has an intermediate radius of gyration. Acetylation of the retained amino-terminal methionine occurred completely with the Ac-Met-Glu-Phe-Leu- and Ac-Met-Asp-Phe-Leu- sequences and partially with the Ac-Met-Asn-Phe-Leu-sequence. Although the consensus for acetylation of the retained amino-terminal methionine is not completely known, these results and the results of published sequences indicated that Ac-Met-Glu- and Ac-Met-Asp- (methionine followed by an acidic residue) is sufficient for amino-terminal acetylation in eukaryotes but not in prokaryotes.     

Yeast methionine aminopeptidase I. Alteration of substrate specificity by site-directed mutagenesis

In eukaryotes, two isozymes (I and II) of methionine aminopeptidase (MetAP) catalyze the removal of the initiator methionine if the penultimate residue has a small radius of gyration (glycine, alanine, serine, threonine, proline, valine, and cysteine). Using site-directed mutagenesis, recombinant yeast MetAP I derivatives that are able to cleave N-terminal methionine from substrates that have larger penultimate residues have been expressed. A Met to Ala change at 329 (Met206 in Escherichia coli enzyme) produces an average catalytic efficiency 1.5-fold higher than the native enzyme on normal substrates and cleaves substrates containing penultimate asparagine, glutamine, isoleucine, leucine, methionine, and phenylalanine. Interestingly, the native enzyme also has significant activity with the asparagine peptide not previously identified as a substrate. Mutation of Gln356 (Gln233 in E. coli MetAP) to alanine results in a catalytic efficiency about one-third that of native with normal substrates but which can cleave methionine from substrates with penultimate histidine, asparagine, glutamine, leucine, methionine, phenylalanine, and tryptophan. Mutation of Ser195 to alanine had no effect on substrate specificity. None of the altered enzymes produced cleaved substrates with a fully charged residue (lysine, arginine, aspartic acid, or glutamic acid) or tyrosine in the penultimate position.     

Receptors for chemotaxis in Bacillus subtilis.

At least three receptors for chemotaxis toward L-amino acids in Bacillus subtilis could be found with the aid of taxis competition experiments. They are called the asparagine receptor, which detects asparagine and glutamine, the isoleucine receptor, which detects isoleucine, leucine, valine, phenylalanine, serine, threonine, cysteine, and methionine, and the alanine receptor, which detects alanine and proline. Histidine and glycine could not be assigned to one of these receptors. Cysteine and methionine were found to be general inhibitors of chemotaxis and serine was found to be a general stimulator of chemotaxis. Some structural analogues of amino acids were tested for chemotactic activity. The chemotactic activity of B. subtilis is compared with that of Escherichia coli.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

Amino Acids as Nitrogen Sources for the Growth of Euglena gracilis and Astasia longa

SYNOPSIS. Euglena gracilis (bacillaris variety, strain SM-L1, streptomycin-bleached) used the following amino adds (10⁻³ M) as sole nitrogen source for growth on a defined medium: glycine, alanine, valine, leucine, isoleucine, serine, threonine, and glutamic acid. Aspartic acid was used at 10⁻² M. Glutamine and asparagine were used at 10⁻³ M and were better N sources than their parent dicarboxylic amino acids. Not used as sole N source for growth were phenylalanine, tyrosine, tryptophan, cysteine, cystine, methionine, proline, hydroxyproline, histidine, arginine, lysine, and taurine. Astasia longa (Jahn strain) was more restricted than Euglena and used only asparagine and glutamine as N sources for growth.     

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.     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

Structures of N-terminally acetylated proteins

Primary structures of 250 characterized proteins with N-terminally acetylated residues were correlated with residue distributions and other data. Excluding multiple forms derived from characterized species variants, the structures represent 105 different types of acetylated proteins. Results of comparisons extend previous suggestions based on fewer structures and define relationships further. The N-terminal residue that is acetylated is of a limited type and is frequently a small residue, with a heavy over-representation of serine and alanine. However, the occurrence of methionine at the acetylated position is also high, whereas that of glycine is less frequent than previously estimated. Lysine is over-represented in the N-terminal region, as is aspartic and glutamic acids at a few positions close to the acetylated N-terminus (especially the adjacent position). Finally, distributions of branched-chain residues in the N-terminal region of acetylated proteins are altered in relation to those of proteins in general, isoleucine is over-represented, and leucine and valine are under-represented. The results suggest that alpha-amino-acetylated proteins have special residues in N-terminally non-hydrophobic structures. Data are compatible with a protective function for acetylation but do not exclude further role(s) in processing or other special functions.     

凤眼莲根分泌物氨基酸组分对根际肠杆菌属F2细菌的趋化作用

凤眼莲(Eichhornia crassipes)的根分泌物中含有Met等多种氨基酸,其中Met、GABA、Gly、Ala、Asp、Ser、Val和Leu(10^-7～10^-2mol·L^-1)均对凤眼莲的根际肠杆菌属F₂(Enterobacter sp.F₂)细菌有强烈的正趋化作用;Glu、Thr和His(10^-7～10^-3mol·L^-1)也对该菌有一定的正趋化作用;而Lys、Cys、Arg、Tyr、Pro、Asn、Gln、Ile、Phe和Typ则对该菌表现出一定的负趋化作用.对细菌的正趋化作用存在一个趋化物的最适浓度范围.具有正趋化作用的氨基酸在凤眼莲根际的浓度都较高,而具有负趋化作用的浓度则较低,这正是凤眼莲与该根际细菌结合为根际微生态系统的原因之一.     

Plasma amino acids in four models of experimental liver injury in rats

Summary We studied the plasma amino acid profiles in four models of hepatic injury in rats. In partially hepatectomized rats (65% of liver was removed) we observed significant increase of aromatic amino acids (AAA; i.e. tyrosine and phenylalanine), taurine, aspartate, threonine, serine, asparagine, methionine, ornithine and histidine. Branched-chain amino acids (BCAA; i.e. valine, leucine and isoleucine) concentrations were unchanged. In ischemic and carbon tetrachloride acute liver damage we observed extreme elevation of most of amino acids (BCAA included) and very low concentration of arginine. In carbon tetrachloride induced liver cirrhosis we observed increased levels of AAA, aspartate, asparagine, methionine, ornithine and histidine and decrease of BCAA, threonine and cystine. BCAA/AAA ratio decreased significantly in partially hepatectomized and cirrhotic rats and was unchanged in ischemic and acute carbon tetrachloride liver damage. We conclude that a high increase of most of amino acids is characteristic of fulminant hepatic necrosis; decreased BCAA/AAA ratio is characteristic of liver cirrhosis; and decrease of BCAA/AAA ratio may not be used as an indicator of the severity of hepatic parenchymal damage.Abbreviations BCAA branched-chain amino acids (i.e. valine, leucine and isoleucine) - AAA aromatic amino acids (i.e. tyrosine and phenylalanine)     

Interaction of chlorambucil with tRNA.

Preincubation of purified mixed tRNAs from Escherichia coli K12-MO with 2.94 mM chlorambucil (CAB) for 2 h at 37 degrees C results in the inhibition of the capacity of mixed tRNAs to accept alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine by 100, 71, 100, 100, 100, 95, 32, 88, 36, 26, 96, 78, 44, 31, 34, 98, 38, and 17% respectively. Preincubation of tRNA with 0.75 mM and 0.29 mM CAB inhibited aminoacylation by aspartic acid to the extent of 69 and 17% respectively. CAB has no apparent effect upon the capacity of ATP to function in the formation of aminoacylated tRNALeu.     

Guanidine hydrochloride induced equilibrium unfolding of mutant forms of iso-1-cytochrome c with replacement of proline-71

Proline-71, an evolutionally conserved residue that separates two short alpha-helical regions, is replaced by valine, threonine, or isoleucine in at least partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae [Ernst, J. F., Hampsey, D. M., Stewart, J. W., Rackovsky, S., Goldstein, D., & Sherman, F. (1985) J. Biol. Chem. 260, 13225-13236]. Treatment of these proteins with a specific sulfhydryl blocking reagent (methyl methanethiosulfonate) to block Cys-102 has allowed investigation of the properties of monomeric forms of the proteins, denoted iso-1-MS. Comparison of the UV-visible absorbance properties (pH 6, 20 degrees C) shows minor differences between the normal Pro-71 iso-1-MS and two of the three mutant proteins. The Val-71 iso-1-MS protein has absorbance properties indistinguishable from those of the normal Pro-71 iso-1-MS protein, but the Ile-71 iso-1-MS and Thr-71 iso-1-MS proteins show reduced intensity of the 695-nm absorbance band and a small shift in the Soret maximum, from 408 nm for the Pro-71 iso-1-MS and Val-71 iso-1-MS proteins to 406 nm for the Thr-71 iso-1-MS and Ile-71 iso-1-MS proteins. Second derivative spectroscopy is used to assess differences in the polarity of the environment of tyrosine residues. The average degree of exposure of tyrosines to solvent is similar in all four proteins: 0.39 for the normal Pro-71 iso-1-MS and Val-71 iso-1-MS proteins; 0.40 for the Ile-71 iso-1-MS protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of the coded amino acids on meso- and ectodermal tissue development in organotypic culture

The effect of L-amino acids was investigated in organotypic tissue culture ofmesodermal tissue (spleen, myocardium) and ectodermal tissue (brain cortex) in mature rats. The low hydrophobic amino acids: asparagine, hystidine, serine, lysine, arginine and glutamine acid, induced the proliferation stimulation. The high hydrophobic amino acids had both the apoptose effect (spleen) and no effect at all (myocardium). The proliferation stimulation occurred in the ectodermal tissue under the effect of the high hydrophobic amino acids (asparagines acid, valine, threonine, methionine, leucine, isoleucine), whereas the low hydrophobic amino acids had no effect on the nervous tissue development. The combination of two amino acids one of which stimulated and another one inhibited the explant growth zone (or was not active in myocardium) lead to an increase of the stimulatory effect in meso- and ectodermal tissue. The amino acid modulated properties can be taken in consideration in synthesis of new regulatory peptides.     

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.     

Metabolic footprinting of the anaerobic bacterium Fusobacterium varium using 1H NMR spectroscopy

Metabolic footprinting of the anaerobic bacterium Fusobacterium varium demonstrated the accumulation of six carboxylic acids as metabolic end-products and revealed specific growth requirements and utilization capabilities towards amino acids. Guided by (1)H NMR determinations of residual amino acids in spent medium, a modified chemically defined minimal medium (CDMM*) was developed by minimizing the amino acid composition while satisfying nutritional requirements to support abundant growth of F. varium. Quantitative determinations of carboxylate salts and residual substrates were readily performed by (1)H NMR analysis of lyophilized residues from CDMM* cultures without interference from initial medium components. Only small concentrations of alanine, arginine, glycine, isoleucine, leucine, methionine, proline and valine were required to support growth of F. varium, whereas larger quantities of aspartate, asparagine, cysteine, glutamine, glutamate, histidine, lysine, serine and threonine were utilized, most likely as energy sources. Both bacterial growth and the distribution of carboxylate end-products depended on the composition of the chemically defined medium. In cultures provided with glucose as the primary energy source, the accumulation of butyrate and lactate correlated with growth, consistent with the regeneration of reduced coenzyme formed by the oxidative steps of glucose catabolism.     

The influence of different nitrogen sources on the production of volatile compounds by Dipodascus aggregatus

Summary The yeast fungus Dipodascus aggregatus was grown aerobically on 9 different nitrogen sources and the production of volatile compounds determined by a gas chromatographic head-space technique. Excellent growth was supported by glutamine, aspartic acid, asparagine, (NH₄)₂-tartrate and NH₄H₂PO₄. Valine, leucine, and particularly isoleucine were utilized with a somewhat lower growth rate. Lysine was rapidly utilized after a prolonged lag phase.The highest production of volatile compounds was obtained from leucine and isoleucine. At least 20 volatile compounds were formed from each of them and many products were detected in high concentrations. Intermediate amounts of volatile compounds were produced from asparagine, the ammonium salts and valine, and low amounts from lysine, glutamine and aspartic acid.Ethyl acetate was a major product irrespective of the nitrogen source used. Regarding the pattern of volatile compounds produced, leucine, isoleucine and valine had much in common. Most of the volatile products formed from these amino acids contained a branched carbon chain and at least three high-boiling components eluted later than n-amyl acetate from the gas chromatographic column. The other six nitrogen sources could be grouped together. In general the same volatile compounds were formed from these sources, but the quantities of the individual compounds differed. Only one component eluted later than n-amyl acetate. No basic difference in production of volatile compounds was observed between the ammonium salts and -amino compounds like lysine and asparagine.     

Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts

The metabolism of ¹⁴C-labeled aspartic acid, diaminopimelic acid, malic acid and threonine by isolated pea (Pisum sativum L.) chloroplasts was examined. Light enhanced the incorporation of [¹⁴C] aspartic acid into soluble homoserine, isoleucine, lysine, methionine and threonine and protein-bound aspartic acid plus asparagine, isoleucine, lysine, and threonine. Lysine (2 millimolar) inhibited its own formation as well as that of homoserine, isoleucine and threonine. Threonine (2 millimolar) inhibited its own synthesis and that of homoserine but had only a small effect on isoleucine and lysine formation. Lysine and threonine (2 millimolar each) in combination strongly inhibited their own synthesis as well as that of homoserine. Radioactive [1,7-¹⁴C]diaminopimelic acid was readily converted into [¹⁴C]threonine in the light and its labeling was reduced by exogenous isoleucine (2 millimolar) or a combination of leucine and valine (2 millimolar each). The strong light stimulation of amino acid formation illustrates the point that photosynthetic energy is used in situ for amino acid and protein biosynthesis, not solely for CO₂ fixation.