Uptake of L-proline by Histoplasma capsulatum.

The uptake and incorporation of L-proline by yeast cells of the dimorphic zoopathogen Histoplasma capsulatum were studied. The amino acid was assimilated in at least two ways: by an active transport system with a Km of 1.7 X 10(-5) M and by simple diffusion. The active transport system was sterospecific and severely restricted to neutral aliphatic side-chain amino acids. Certain analogues inhibited L-proline uptake and prevented incorporation of the amino acid into cellular constituents. The inhibition of L-proline uptake by L-leucine was competitive. Since L-leucine and L-proline are seemingly transported by a system with similar characteristics, must be concluded, as originally postulated, that the buckled ring of L-proline, in solution, acts as an aliphatic side chain and that this cyclic amino acid is transported by a system more or less specific for amino acids with neutral aliphatic side chains.     

E.s.r. of spin-trapped radicals in gamma-irradiated polycrystalline DL-alanine. A quantitative determination of radical yield

The quantitative aspects of determining free radicals in polycrystalline amino acids gamma-irradiated at room temperature and subsequently dissolved in spin-trap solutions were investigated. The deamination radical in DL-alanine was used for detailed studies and 2-methyl-2-nitrosopropane (MNP) was employed as the spin-trap. The spin-trapping efficiency (the number of radicals spin-trapped in solution divided by the number of radicals initially present in the gamma-irradiated solid) was found to be in the range 1 to 10 per cent for aqueous solutions depending on the experimental conditions. The effects of dose, particle size, pH, spin-trap concentration, age of spin-trap solution, MNP monomer to dimer ratio and the presence of organic solvents were investigated. Several reactions were found to decrease the spin-trapping efficiency; radical-radical recombination, the competition between the spin-adduct and the spin-trap for radicals and the reaction of radicals with the MNP dimer. The reaction of intact DL-alanine molecules with deamination radicals to produce H-abstraction radicals which are not spin-trapped does not significantly lower the spin-trapping efficiency. The results obtained with compounds such as glycine, glycylglycine, L-valine and L-proline suggest that the low spin-trapping efficiency found for DL-alanine may be representative of polycrystalline amino acids.     

Interactions between the branched-chain amino acids in the growth of Spirodela polyrhiza

The joint action of L-valine and L-isoleucine, L-leucine and L-isoleucine, and L-valine and L-leucine on the growth of Spirodela polyrhiza was established. The effect of one branched-chain amino acid on growth inhibition by another one was compared with the non-specific antagonisms which glycine and L-alanine exert on growth inhibition by singly supplied branched-chain amino acids. In this way specific and non-specific interactions could be distinguished. It appeared that: (1) L-isoleucine was a specific antagonist of L-valine; (2) L-leucine was a specific antagonist of L-isoleucine; (3) L-valine and L-leucine were synergistic growth inhibitors. Further, it was found that: (4) growth inhibition by L-leucine was specifically antagonized by simultaneously supplied L-valine and L-isoleucine; (5) an excess of L-isoleucine strongly inhibited the conversion of exogenous valine into leucine; (6) accumulation of valine was typical of isoleucine-induced growth inhibition. The results are consistent with the view that growth inhibition by L-valine and L-leucine is due to the blocking of acetohydroxy acid synthetase, the first common enzyme in the valine-isoleucine biosynthetic pathway. Growth inhibition by L-isoleucine, however, seems to result from inhibition of leucine synthesis at a step after 2-oxoisovaleric acid. Some aspects of the regulation of branched-chain amino acid biosynthesis in higher plants are discussed.     

Synthesis, spectroscopic, mutagenic, and cytotoxicity studies of some mixed-ligand platinum(II) complexes of 2,2'-bipyridine and amino acids

Seven platinum(II) complexes of the type [Pt(bipy)(AA)]n+ (where n = 1 or 0 and AA is anion of L-valine, L-isoleucine, L-aspartic acid (dianion), L-glutamic acid (dianion), L-glutamine, L-proline, or S-methyl-L-cysteine) have been prepared and characterized. The modes of binding of amino acids in these complexes have been ascertained particularly by infrared and 1H NMR spectral studies. The L-glutamine complex shows a ID50 value (50% inhibitory dose) in the range of greater than 20 micrograms/ml to 100 micrograms/ml of the complex. However, the complexes of L-valine, L-isoleucine, L-aspartic acid, L-glutamic acid, L-proline, and S-methyl-L-cysteine show ID50 values greater than 100 micrograms/ml of the complex. The above complexes also show inferior growth inhibition of P-388 cells than platinum(II) complexes of 2,2'-bipyridine with L-alanine, L-leucine, L-methionine, and L-aspargine as reported earlier. The platinum(II) complexes of 2,2'-bipyridine with glycine (Gly), L-alanine (Ala), L-leucine (leu), L-valine (Val), L-methionine (Met), L-phenylalanine (Phe), L-serine (Ser), L-tyrosine (Tyr) and L-tryptophan (Trp) have been tested for mutagenesis using TA 100 and TA 98 strains. They show nonmutagenicity. This is in contrast to the cis-[Pt(NH3)2Cl2] showing a base pair substitution mutagenesis.     

Proline uptake by monolayers of human intestinal absorptive (Caco-2) cells in vitro.

Monolayers of the Caco-2 human intestinal cell line exhibit active and passive uptake systems for the imino acid L-proline. The active transport component is saturable and it is responsible for about two thirds of the observed flux over the nanomolar concentration range, at 37 degrees C and pH 7.4. In contrast to L-phenylalanine, specific L-proline uptake has a high degree of sodium dependency and the efficiency of the carrier system is significantly reduced when protein synthesis (cycloheximide), Na+/K(+)-ATPase (ouabain) or cellular metabolism (sodium azide) are inhibited. The expression of the L-proline carrier by Caco-2 cells was under some degree of nutritional control. Glucose deficiency, over the time scale of the experiment, had no effect. The temperature-dependence of the specific uptake process followed the Arrhenius model with an apparent activation energy of 93.5 kJ nmol-1. This pathway also displayed Michaelis-Menten concentration-dependence with a Ksdm of 5.28 mM and a maximal transport flux (Jsdmax) of 835 pmol min-1 (10(6) cells)-1. Although the passive component was unchanged, the pH of the donor phase exerted a profound effect on the active carrier component. Within the physiological pH range a local maximum efficiency was found at pH 7.4 but dramatic increases were noted as pH 5.0 was approached. In competition studies, with 100-fold excess of a second amino acid, strong inhibition of uptake was found with alpha-aminoisobutyric acid, L-alanine and L-serine whereas moderate inhibition was observed with glycine, D-proline and gamma-aminoisobutyric acid. Aromatic and branched amino acids showed weak (L-valine) or no interaction (L-phenylalanine, L-leucine) with the carrier system. These data indicate that the carrier system for the uptake of L-proline has many features in common with the A system for amino acid transport.     

Behavioral responses of the brittle star Ophiura ophiura to chemical stimuli during adaptation of amino acid chemoreceptors

Parts of the feeding behavior, the orienting movements and the‘walking’ behavior pattern in the brittle star Ophiuraophiura are released by several low-molecular-weight compounds.The behavioral responses of this animal were adapted to 10^–4molar concentrations of the following single stimulatory compounds:L(+;)lactic acid, glycine, sarcosine, L-alanine, L-cysteine,L-proline, L-valine, L-leucine, L-arginine and acetylcholineiodide. Complete adaptation of the behavioral response for theentire experimental group of animals occurred for most chemicalswithin the fifth to the ninth minute after the introductionof the adapting stimulus; however, for acetylcholine iodide,the time to behavioral adaptation was up to 15 min. The sameten and several other compounds were tested during each of theadaptations. Test concentrations were calculated to be below3 x 10^–5 molar at the animals' arms. Narrowly-tuned receptorsites were indicated for L-valine, L-leucine, L-arginine andacetylcholine iodide since behavioral responses to these chemicalswere not crossadapted by any of the other amino acid stimulitested. The ‘walking’ responses to L-proline, L-cysteineand L-alanine, which were the least effective substances tested,were cross-adapted by the majority of the adapting stimuli.Glycine and sarcosine did not cross-adapt the responses to eachother which indicated independent receptors for these stimuli.Independent receptor sites located on separate receptor cellswere suggested for L-proline, which stimulated the arm coilingresponse at high concentrations, and for ß-alanineand thioglicolic acid, which stimulated the tube feet walkingbehavior.     

Conversion of ammonia or urea into essential amino acids, L-leucine, L-valine, and L-isoleucine, using artificial cells containing an immobilized multienzyme system and dextran-NAD+. 2. Yeast alcohol dehydrogenase for coenzyme recycling.

Semipermeable nylon-polyethylenimine artificial cells containing leucine dehydrogenase (EC 1.4.1.9), alcohol dehydrogenase (EC 1.1.1.1), urease (EC 3.5.1.5), and dextran-NAD+ were prepared. Artificial cells could convert ammonia or urea into L-leucine, L-valine, and L-isoleucine. For batch conversion in 20.0 mM of ammonium acetate substrate solutions, in 2 h 0.2 ml of artificial cells could produce 4.48 mumol of L-leucine, 9.98 mumol of L-valine, or 5.96 mumol of L-isoleucine. The corresponding conversion ratios were 22.4, 49.9, and 29.8%. In 20.0 mM of urea substrate solutions, 13.71 mumol of L-leucine, 16.12 mumol of L-valine, or 13.44 mumol of L-isoleucine was produced and the conversion ratios were 68.6, 80.6, and 67.2%. The substrate specificity of leucine dehydrogenase for the reductive amination was determined. Of the three branched-chain amino acids produced, the production rates of L-valine were the highest. The apparent Km values were as follows: 0.32 mM for alpha-ketoisocaproate, 1.63 mM for alpha-ketoisovalerate, and 0.73 mM for Dl-alpha-keto-beta-methyl-n-valerate. The leucine dehydrogenase multienzyme system had a good storage stability. It retained 72.0% of the original activity with artificial cells were stored at 4 degrees C for 6 weeks. The optimum conversion pH and temperature were 8.5-9.0 and 35-40 degrees C. The effects of urea and ammonium salts on conversion rate were also studied. The relative activities in ammonium salts solutions were 45.1-75.9% of those in urea solutions.     

Solvent-free crystallizations of amino acids: the effects of the hydrophilicity/hydrophobicity of side-chains

This work studied the self-assembling (crystallizing) behaviour of amino acids in the absence of solvent and additives (by sublimation and deposition in vacuum), instead of from aqueous solution. It is found that the hydrophilicity/hydrophobicity of side-chains can significantly affect the crystallization of amino acids in the absence of solvent. Crystal structures of amino acids having hydrophobic side-chains (L-valine, L-leucine, L-isoleucine and l-methionine) obtained from sublimation are the same with those obtained from aqueous solution. New polymorphs for six amino acids are thought to have been obtained, based on X-ray diffraction and IR data for three of them (L-tyrosine, L-Phyenylalanine and L-tryptophan), and just IR data for the other three (L-alanine, L-proline and L-threonine).     

A comparative study of essential arginine residues in Gramicidin S synthetase 2 and isoleucyl tRNA synthetase

In gramicidin S synthetase 2 (GS 2) from Bacillus brevis, L-proline, L-valine, L-ornithine, and L-leucine activations to aminoacyl adenylates are progressively inhibited by phenylglyoxal. The inactivation of GS 2 obeys pseudo-first-order kinetics. ATP completely prevents inactivation of GS 2 by phenylglyoxal, whereas amino acids only partially prevent it. In the presence of ATP, four arginine residues per mol of GS 2 are protected from modification by phenylglyoxal as determined by amino acid analysis and the incorporation of [7-14C]phenylgloxal into the enzyme protein, indicating that a single arginine residue is necessary for each amino acid activation. In isoleucyl tRNA synthetase from Escherichia coli, phenylglyoxal inhibits activation of L-isoleucine to isoleucyl adenylate. ATP completely prevents inactivation, although isoleucine only partially prevents it. One arginine residue of isoleucyl tRNA synthetase is protected by ATP from modification by phenylglyoxal, suggesting that a single arginine residue is essential for isoleucine activation. These results support the involvement of arginine residues in ATP binding with GS 2 or isoleucyl tRNA synthetase, and thus indicate that arginine residues of amino acid activating enzymes are essential for the formation of aminoacyl adenylates in both nonribosomal and ribosomal peptide biosynthesis.     

Far-infrared spectra of sequential polypeptides with -helical and polyglycine II structures

The sequential copolymers of glycine and L -alanine, L-valine and L -alanine, L-leucine and L -alanine, and L-phenylalanine and L -alanine and those containing the L-proline residues were synthesized. The infrared spectra in the region from 700 to 200 cm^-1 were measured for these polypeptides with the α-helical conformation or the polyglycine II structure and compared with the spectra of the β-form structures. The results showed that several infrared bands observed in the region from 600 to 200 cm^-1 clearly reflect not only the backbone conformations but also the local conformations of component amino acid residues of polypeptides with the α-helical, β-form and polyglycine II structures.     

Characterization and location of the L-proline activating fragment from the multifunctional gramicidin S synthetase 2

Gramicidin S synthetase 2 (GS2) derived from Bacillus brevis is a multifunctional single polypeptide (Mr 280,000) with a 4'-phosphopantetheine residue covalently bound to the enzyme. When GS2 was treated with trypsin or chymotrypsin, fragments with some activity were liberated. The molecular mass of the L-proline activating fragment was 114 kDa on SDS-PAGE. This fragment, when incubated with gramicidin S synthetase 1 (GS1) in the presence of phenylalanine and proline, produced D-Phe-L-Pro dipeptide. The fragment accepted D-phenylalanine from GS1 in the absence of L-proline. The L-proline activating fragment was shown to lack pantothenic acid by microbiological assay. On the other hand, the L-leucine activating fragment, which was partially purified, contained a large amount of pantothenic acid, although it did not form the D-Phe-L-Pro dipeptide. These results indicate that the L-proline activating site is located near an acceptor site for D-phenylalanine on GS2, but that it is not adjacent to a 4'-phosphopantetheine group. The N-terminal sequence (15 amino acid residues) of the L-proline activating fragment obtained by trypsin treatment was identical with that of GS2, indicating that the L-proline activating site is located at the N-terminus of the native synthetase. The N-terminal sequence of GS2 has been matched with the amino acid sequence deduced from the nucleotide sequence 71 bp downstream of the stop codon of the GS1 gene except that the first initiator methionine was not detected.     

E.s.r. of spin-trapped radicals in aqueous solutions of amino acids. Reactions of the hydroxyl radical.

The radicals produced by reactions of hydroxyl radicals with amino acids in aqueous solutions have been investigated. Hydroxyl radicals were formed by U.V.-photolysis of hydrogen peroxide and the short-lived amino acid radicals were spin-trapped by tert-nitrosobutane and identified by electron spin resonance spectroscopy. Nineteen amino acids were studied, and several radicals were identified which have not been observed previously by other methods. Only side-chain radicals were identified for alanine, threonine, aspartic acid, asparagine, lysine, phenylalanine, tyrosine, proline and hydroxyproline; whereas for glycine the C(2) carbon radical was spin-trapped. Both C(2) carbon radicals and side-chain radicals were assigned to valine, leucine, isoleucine, serine, glutamic acid, glutamine, arginine and methionine.     

四种氨基酸原料细菌内毒素检查法的研究

依据中国药典2000年版“细菌内毒素检查法”,通过鲎试剂的干扰试验,研究注射用氨基酸原料中细菌内毒素检查法的可行性。结果在2~16倍稀释级下,L-缬氨酸(c=2.5%)和L-异亮氨酸(c=2.5%)对鲎试剂无干扰,检测细菌内毒素的鲎试剂灵敏度≤5.0×102EU.L-1,而在16倍稀释级范围内,L-脯氨酸(c=4.5%)和L-亮氨酸(c=2.0%)对鲎试剂检查法有抑制作用。结论为注射用L-异亮氨酸和L-缬氨酸用灵敏度为5.0×102EU.L-1的鲎试剂检查其细菌内毒素方法可行,可以代替家兔法检查热原。     

Behavioral study of chemoreception in the sea starMarthasterias glacialis: Structure-activity relationships of lactic acid,amino acids,and acetylcholine

Behavioral responses of Marthasterias glacialis to low molecular compounds were studied under laboratory conditions. Feeding postures, stomach eversions and locomotion of initially inactive animals can be released with very dilute solutions of lactic acid, neutral 2 and 3 carbon amino acids, L isomers of 4 to 6 carbon neutral amino acids, L-arginine, acetylcholine iodide, and several of their analogues. Hunger was induced by temporary withdrawal of food. Responsiveness to feeding stimuli was controlled with L-cysteine and L-leucine. The lowest behavioral thresholds for the most effective feeding stimuli were 3 X 10(-11) mol/l for both enantiomers of lactic acid, 10(-8) mol/l for L-proline and both enantiomers of cysteine and 10(-7) mol/l for acetylcholine iodide and some of the effective neutral amino acids. The behavioral threshold values for chemical stimuli differed by a factor between 30 and 100 in different sea stars. The test concentration was 3 X 10(-7) mol/l, the level at which L-cysteine elicited a complete feeding response from all the animals. Structure-activity comparison of substances less effective than the control stimulus was thus possible. The behavioral threshold of fully effective substances was determined later. The independence of receptor mechanisms for different substances can be inferred as: L-cysteine controlled responsiveness is not always accompanied by responsiveness to neutral amino acids. Autotomized marthasterias arms crawled after stimulation with lactic acid, cysteine, and acetylcholine iodide but did not respond to the feeding stimuli betaine and L-proline. An animal became inactive if electric shocks were paired with L-proline or L-cysteine emanating from an 'electric' food model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spin trapping of lipid-derived radicals in liposomes

Electron-spin resonance-spin trapping has been used to detect lipid-derived radicals in liposomes. Using the lipid-soluble spin trap 2-methyl-nitrosopropane (MNP), we have detected both the lipid and hydrogen-atom spin adducts in liposomes composed of a fully saturated phospholipid (dimyristoylphosphatidylcholine, DMPC) with various mol fractions of unsaturated phospholipid (1-palmitoyl-2-arachidonoylphosphatidylcholine, PAPC) or fatty acid (arachidonic acid, AA). The lipid-derived spin adduct formed during autoxidation of liposomes was separated by thin-layer chromatography and found to co-migrate with the product(s) formed by direct addition of MNP to the corresponding unsaturated lipid or fatty acid. Both the MNP-PAPC and MNP-AA spin adducts showed some restriction of rotational motion when in the liposome bilayer (rotational correlation times 0.72 and 0.69.10(-9) s, respectively), and nitrogen hyperfine coupling constants (14.94-14.96 G) consistent with a hydrophobic localization. Radical versus non-radical mechanisms of spin adduct formation during liposome autoxidation were separated using alpha-tocopherol as a radical scavenger. The utility of nitroso spin traps in trapping of radicals in liposomes is discussed.     

Evidence that cycloleucine affects the high-affinity systems of amino acid uptake in cultured human fibroblasts.

The influence of cycloleucine on kinetic parameters of uptake of L-alanine, L-proline and L-leucine into cultured human fibroblasts was examined under initial-rate conditions with substrate concentrations of 0.05-10 mM and 5 mM-cycloleucine. Kinetic data obtained by computer analysis showed that, in the absence of cycloleucine, cell uptake was heterogeneous for each amino acid. L-Alanine and L-leucine entered by two transport systems with different affinities; L-proline was taken up by one saturable transport system plus a diffusion-like process. This heterogeneity disappeared in the presence of cycloleucine, since the high-affinity systems were no longer detectable. The remaining process had the same kinetic constants as the low-affinity system for alanine and leucine and a KD similar to the diffusion constant for proline. The influence of cycloleucine on the amino acid uptake was not specific either to the amino acid concerned or to a particular transport system, since the three neutral amino acid-transport systems, A, ASC and L, were involved in these experiments. This influence was shown to be unaffected by the absence of Na+ (for leucine uptake). ATP content of the cells was identical in the presence or in the absence of cycloleucine.     

Ternary complexes of palladium (II) with levamisole and L-amino acids

The complexes of general formula [(LMS)2Pd(amino acid)]Cl with LMS = levamisole, and amino acid = L-alanine, L-phenylglycine, L-phenylalanine, L-valine, L-methionine, and L-proline, were synthesized by the interaction of [(LMS)2PdCl2] with the sodium salts of L-amino acids. The newly synthesized complexes are characterized by elemental analysis, conductivity, magnetic susceptibility, optical rotation measurements, and UV-Vis, IR and 13C NMR spectral data. Levamisole is coordinated to palladium via the N-7 nitrogen and the amino acids through the amino nitrogen and carboxylate oxygen, except for L-methionine which binds the metal via nitrogen and sulfur atoms. Optically active [(LMS)2Pd(amino acid)]Cl complexes are obtained when L-amino acids or D,L-amino acids are used for the synthesis of these complexes. L-Methionine and L-proline complexes induce new cell forms in Baker's yeast (Saccharomyces cerevisiae) cells.     

Defined minimal media for the growth of prototrophic and auxotrophic strains of Bacillus stearothermophilus

Minimal chemically defined media for Bacillus stearothermophilus were developed at 60°C and quantitative requirements for each nutrient were determined. A prototrophic strain of B. stearothermophilus was grown in medium containing only glucose and mineral salts whereas auxotrophic strains in addition required biotin, thiamine, nicotinic acid and DL-methionine. Metabolic interaction between L-valine and L-leucine was observed with auxotrophic organisms. The presence of L-leucine in minimal medium necessitated the addition of L-valine. Growth took place in the absence of both amino acids.     

Studies on the regulation of leucine catabolism in the liver. Stimulation by pyruvate and dichloroacetate

The rate of oxidation of L-[1-14C]leucine to 14CO2 by isolated rat hepatocytes is increased by pyruvate and dichloroacetate. This effect is specific for L-leucine, not being observed for L-valine, L-isoleucine, or D-leucine. Transamination, the rate-limiting step of L-leucine catabolism in the liver, is the site of stimulation, because uptake of L-leucine by the cells and the oxidation of its transamination product, alpha-ketoisocaproate, are not increased. Measurement of steady state levels of alpha-ketoisocaproate indicate that both pyruvate and dichloroacetate promote the transamination of L-leucine, thereby increasing the availability of substrate for decarboxylation by the alpha-ketoisocaproate dehydrogenase complex (EC 1.2.4.3). Pyruvate stimulation of transamination is secondary to the provision of keto acid acceptors for the amino group of L-leucine. The mechanism of the effect of dichloroacetate remains unknown.     

Production of Nucleic Acid-related Substances by Fermentative Processes: XIX. Accumulation of 5′-Inosinic Acid by a Mutant of Brevibacterium ammoniagenes

The accumulation of 5′-inosinic acid (IMP) by a mutant, KY 13102, induced from Brevibacterium ammoniagenes ATCC 6872 by ultraviolet light irradiation, was examined. Although growth was stimulated by adenine or adenosine, the microorganism showed fair growth in the medium containing amino acids but no adenine. Among six kinds of natural nutrients tested, meat extract and Casamino Acids were suitable for the accumulation of IMP. Manganese ion strongly affected growth, the accumulation of IMP and hypoxanthine, and cell morphology. Among amino acids tested, L-methionine, L-proline, and L-valine stimulated IMP accumulation. In the medium containing 1.0 g of L-proline per liter, 12.8 mg of IMP per ml was accumulated. The mechanism of IMP accumulation by the mutant is discussed.