Far-infrared spectra of copoly(- -amino acids)

Far-infrared spectra in the region from 700 to 200 cm^?1 were measured for the copolymers of L -alanine and glycine, those of L -alanine and L -valine, those of L -alanine and L -leucine, and those of L -alanine and L -phenylalanine. The observed spectra were interpreted on the basis of the analysis of the far-infrared spectra of the corresponding homopolymers, and the correlation between the conformations of the copolymers and the kinds of the component amino acids was discussed.     

The random copolymerization of γ-benzyl-L-glutamate and L-valine N-carboxyanhydrides: Reactivity ratio and heteogeneity studies

The random copolymerization of the N-carboxyhydrides of γ-benzyl-L -glutamate and L -valine using triethylamine as the initiator in low dielectric media reults in high-molecular-weight copolymers at low convenrson. This behavior makes it possible to apply the monomer reactivity ration theory, which was dervied for addition polymerizations, and from the use of the copolymer composition equation, the respective monomer reactivity ratios, the average and incremental copolymer compositions, and the monomer feed ratio at any conversion can be determined. A comparison of the reactivity ratios for the copolymerization of γ-benzyl-L -glutamate NCA and L -valine NCA in benzene/methylene chloride (r_G = 2.1, r_V = 0.6) with those obtained using dioxane (r_G = 2.7, r_V = 0.3) indicates that the interchain compositional heterogeneity is greater for copolymers prepared in the dioxane. For Example, at 100% conversion of the monomeric NCAs, Poly[Glu(OBzl)⁵⁰Val⁵⁰] prepared in dioxance has an interchain composition ranging from 74 to 0 mol % γ-benzyl-L -glutamate, whereas in benzene/methylene chloride the interchain composition of γ-benzyl-L -glutamae ranges from 65 to 0 mol %. Once the reactivity ratios are obtained for any pair of α-amino and N-carboxyanhydrides, the use of the aforementioned parameters relating to interchain composition can give insight into the compositional heterogeneity between chains as a function of conversion and provide a basis for the preparation of random α-amino acid copolymers that are homogeneous.     

Oligonucleotides containing a peptide internucleotide linkage. A novel class of modified oligonucleotides

Oligonucleotide analogues containing one or a few glycine, L-, and D-alanine residues instead of phosphodiester internucleotide linkages were synthesized (C3′-NH-C(O)-CH(X)-NH-C(O)-C4′, where X = H, (S)-CH₃, and (R)-CH₃. The stability of the duplexes of modified oligonucleotides with their wild-type complements was studied. The incorporation of glycine and L-alanine residues into internucleotide linkages was shown to noticeably decrease the stability of modified duplexes as compared to that of native ones (ΔT _m∼−2°C per modification), whereas analogues containing D-alanine linkers form duplexes with increased stability (ΔT _m∼+2°C per modification).     

Heat capacities of solid poly(amino acid)s. II. The remaining polymers

In the first paper heat capacities C_p, of polyglycine, poly(L -alanine), and poly (L -valine) were analyzed using approximate group vibrations and fitting the C_p contributions of the skeletal vibrations to a two-parameter Tarasov function. In this second paper all other poly (amino acid) s are similarly analyzed. Heat capacities were measured by differential scanning calorimetry in the temperature range of 230–390 K for poly(L -leucine), poly(L -serine), poly (sodium-L -aspartate), poly(sodium-L -glutamate), poly(L -asparagine), poly(L -phenylalanine), poly(L -tyrosine), poly(L -methionine), poly (L -tryptophane), poly(L -proline), poly(L -lysine · HBr), poly(L -histidine), poly(L -histidine- HCl), and poly(L -arginine · HCl). Good agreement exists between experiment and calculation. Predictions of heat capacities were made for all not-measured poly (amino acid) s. Enthalpies, entropies, and Gibbs functions for the solid state have been derived. © 1993 John Wiley & Sons, Inc.     

Correlation between the sequence-length distribution and structure of statistical copolymers of L-valine and γ-benzyl-L-glutamate

Statistical copolymers were prepared from N-carboxyanhydrides of L -valine and γ-benzyl-L -glutamate in dioxan with triethylamine as an initiator. The copolymerization conversion was determined by ir spectroscopy, the copolymer composition by amino acid analysis, and the molecular weights by light scattering. The monomer reactivity ratios were found to be r_Val = 0.14 and r_Glu(OBzl) = 6.4. High-molecular-weight copolymers are formed even at low conversions. The content of β-structure in the copolymers was estimated from the ir spectra in copolymerization mixtures. The sequence-length distribution of L -valine and γ-benzyl-L -glutamate copolymers was calculated and its dependence on copolymerization conversion is discussed. Relations between the sequence-length distribution and the content of β-structure were studied. It was found that the content of β-structure in samples with the same composition is different for low- and high-conversion copolymers. The formation of β-structure in copolymers in the copolymerization mixture requires a certain minimal sequence length, which has been found to be about 6 valine units.     

Far-infrared spectra of sequential copolymers of amino acids with alkyl group side chains

Far-infrared spectra were measured for the sequential copolymers of amino acids with alkyl group side chains. The analysis of the spectra showed that (L -Ala-L -Ala-Gly)_n, (L -Ala-Gly)_n, (L -Ala-Gly-Gly)_n, (L -Val-L -Ala-L -Ala)_n, and (L -Val-L -Ala)_n, have the antiparallel pleated sheet structures and that the backbone conformations of (L -Val-L -Val-L -Ala)_n and (L -Val-L -Val-Gly)_n are the same as that of poly-L -valine. The far-infrared bands characteristic of the antiparallel pleated sheet structure were assigned on the basis of the result of the normal coordinate analysis of poly-L -alanine with this structure. The intersheet and interchain spacings of the sequential copolymers with the antiparallel pleated sheet structure were determined from the x-ray powder-diffraction patterns of these samples.     

Conformations of poly-L-valine in solution

In order to test theoretical predictions that poly-L -valine can exist in an α-helical conformation, water-soluble block copolymers of L -valine and D , L -lysine were prepared. By carrying out the synthesis on a resin support (with the use of N-carboxyanhydrides) contamination of the individual blocks by any unreacted monomer from the previous block was avoided. A single glycine residue was incorporated at the C-terminus of the chain for use in amino acid analyses. Using optical rotatory dispersion and circular dichroism criteria, about 50% of the short valine block of (D , L -lysine HCl)₁₈-(L -valine)₁₅-(D , L -lysine-HCl)1₆-glycine was found to be in the right-handed α-helical conformation in 98% aqueous methanol, in water, the polymer appears to be a dimer, with the valine block being involved in the formation of an intermolecular β-structure.     

Kinetics of the reactions of pentacyanonitrosylferrate(II) with mono- and diamino acids

The reaction of Fe(CN)₅NO^2? with glycine, α-alanine, β-alanine, γ-aminobutyric acid, ornithine and lysine were gas-volumetrically studied in a weakly-alkaline medium. The kinetic data show that the reactivity of the amino group depends on the basicity of the amine, on the behaviour of nucleophilic centers of the carbon chain, and on their steric positions.The kinetic results are compared to the data of the reactions of amino acids with nitrous acid and of the complex with aliphatic amines.     

Synthèse et structure de la poly-O-carbobenzoxy L- tyrosine et de copolymères de O-carbobenzoxy L-tyrosine et de glutamate de benzyle ou d'aspartate de benzyle

The optical rotatory dispersion of copolymers of O-carbobenzoxy-L -tyrosine and benzyl L (or D )-glutamate as well as benzyl L -aspartate, dissolved in nonpolar solvent, has been studied. Moffitt's equation permits the determination of b₀ coefficients whose variation, with varying composition in amino acid residues, suggests that the molecules of poly-O-carbobenzoxy-L -tyrosine have a helical structure similar to that of poly-(benzyl L -glutamate). Results obtained from infrared spectroscopy and x-ray diffraction show that the copolymers possess a helical conformation in the solid state, even when they are very rich in carbobenzoxy-L -tyrosine residues. The value of the b₀, coefficient for poly-O-carbobenzoxy-L -tyrosine may be explained by a regular stacking of the chromophore groups around the helical backbone. The ordering of the molecules of this polymer in a purely helical structure seems favored by the insertion of a small number of foreign residues in the polypeptide chain.     

Structure of Rugulovasine A,B and their Derivatives

Culture conditions for the preparation of cells containing high tyrosine phenol lyase activity were studied with Erwinia herbicola ATCC 21434. Adding pyridoxine to the medium enhanced enzyme formation, suggesting that it was utilized as a precursor of the coenzyme, pyridoxal phosphate. Glycerol plus succinic acid; amino acids, such as, DL-methionine, DL-alanine and glycine; and metallic ion, ferrous ion promoted enzyme formation as well as cell growth. Adding L-tyrosine, as inducer, to the culture medium was essential for enzyme formation. However, when large amounts of L-tyrosine were added, the enzyme formation was repressed by the phenol liberated from L-tyrosine. In fact, formation of the enzyme was enhanced by removing phenol during cultivation. L(D)-Phenylalanine or phenylpyruvic acid had a synergistic effect on the induction of enzyme by L-tyrosine.

Cells with high enzyme activity were prepared by growing cells at 28°C for 28 hr in a medium containing 0.2% L-tyrosine, 0.2% KH₂PO₄, 0.1% MgSO₄7H₂O, 0.001% FeSO_4·7H₂O, 0.01% pyridoxine-HC1, 0.6% glycerol, 0.5% succinic acid, 0.1% DL-methionine, 0.2% DL-alanine, 0.05% glycine, 0.1% L-phenylalanine and 120 ml/liter hydrolyzed soybean protein in tap water with the pH controlled at 7.5 throughout cultivation.     

Biochemical Studies on Germination of Bacterial Spores

The initiating mechanism in the germination of Bacillus thiaminolyticus spores was studied with ¹⁴C-L -alanine. A characteristic pattern of incorporation of L -alanine into the spores was observed during the early stages of germination with two incorporation peaks, one occurred just after contact with L -alanine (first incorporation) and the other 5 min later (second incorporation). L -Glutamine, L -valine, or L -serine substituted for the incorporation of L -alanine during the first stage of germination. Although, L -alanine taken up during the first incorporation phase was extractable with trichloroacetic acid (TCA), that taken up during the second incorporation phase was not extractable. The distribution of radioactivity showed that incorporated L -alanine was located in the spore coat, mainly in the paracrystal fraction. The radioactive material which remained in the germination medium or was extractable from the spore coat fraction with TCA treatment or pronase digestion was identified as alanine. Significance of incorporation of L -alanine and its location in the spore in reference to the initiation of germination is discussed.     

Poly(L-valine). Conformational dependence on the degree of polymerization: infrared studies.

The infrared spectra of poly(L -valine)'s with varying degrees of polymerization have been investigated, as well as copolymers of L -alanine and L -valine. The spectra of nujol mulls of various molecular-weight poly(L -valine)'s, isolated directly from the polymerization media, as well as spectra of these same samples after treatment with strong acid, are recorded. In the 700–250-cm^?1 region, bands at 543 and 414 cm^?1 are found to increase with increasing degree of polymerization in the nujol mulls, but are missing in the acid-treated samples. These bands are assigned to the L -valine residues with an β-helixlike local conformation. It is inferred that the polymerization proceeds initially in the β form, and after a critical degree of polymerization the chains adopt an appreciable amount of an α-helixlike local conformation.     

Susceptibility behaviour and specific heat anomaly in single crystals of alanine and valine

Magnetization of single crystals ofD-,L-alanine andD-valine were measured as a function of temperature using the SQUID magnetometer. An obvious lambda transition at 270 ± 1 K was shown in the specific heat measurement of alanine and valine enantiomers by differential scanning calorimetry (DSC). Magnetic transition temperature seems coincident with that of lambda transition. Temperature dependence of the X-ray powder diffraction forD-valine showed no crystal lattice change under the temperature cooling down from 293 K to 123 K. We propose that the difference of theX T curve between theD-alanine andL-alanine is attributable to the variation of intramolecular geometry of chirality density in the presence of an external magnetic field. The chirality characteristics of alanine and valine enantiomers by the specific heat and susceptibility behaviour are discussed.     

Association studies of N-acetyl-amino acid N,N-dimethylamides in carbon tetrachloride

The self-association of N-acetylglycine N,N-dimethylamide, N-acetyl-L -valine N,N-dimethylamide, and N-acetyl-L -phenylalanine N,N-dimethylamide in carbon tetrachloride was investigated by using ir and ¹H-nmr methods. It was concluded from ir measurements that the associated species is the dimer formed as a result of the simultaneous formation of two intermolecular hydrogen bonds. This is supported by the results of ¹H-nmr measurements. Thermodynamic quantities for the association were determined from the temperature and concentration dependence of the NH proton chemical shifts of the sample solutions. Compared with the Gly derivative, L -Val and L -Phe derivatives have larger values of ?ΔH for association, which shows good correlation with Δv_NH values, the difference between the maxima of the monomer and dimer bands, obtained from ir spectra. This is due to the less stable monomer conformation and to the stronger intermolecular hydrogen bonding of the dimers in L -Val and L -Phe derivatives. The line shapes of both methyl proton resonances of L -Val residue and methylene proton resonances of L -Phe residue were found to vary with concentration and temperature of the sample solutions. These data indicate that the rotation about the C^α—C^β bond is restricted by the steric hindrance present in the associated dimers. All these experimental results can be related to the fact that L -Val and L -Phe derivatives have a warped framework because of the bulky side chains, whereas the Gly derivative has a planar framework.     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Block sequential polypeptides of L-alanine and glycine with D, L-glutamic acid

Sequential polypeptides of L -alanine(A) and glycine(G), which were incorporated between two blocks of poly(D ,L -glutamic acid) (DL), were synthesized by applying Merri-field's solid-phase method. On the basis of optical rotatory dispersion criteria, DL(A)₃₈-DL was found to assume the α-helix in the whole range of the water-methanol system; whereas other block sequential polypeptides were found to assume the random-coiled conformation in water and partly the α-helix at the high methanol content. The stability of the α-helix decreased in the order: DL(A)₃₈DL, DL(A₂G)₁₀DL, DL(A₂G)₆DL, and DL(A₃G)₇DL. This phenomenon may be explained in terms of the dependence of hydrophobic bonding between the C₃H group of the ith L -alanine regularly arranged on the surface of the α-helix and the C₂H group of the (i + 3)th residue on whether the residue is alanine or glycine. The role which the methanol plays in stabilizing the α-helix is also discussed.     

l-Valine production with minimization of by-products’ synthesis in Corynebacterium glutamicum and Brevibacterium flavum

Corynebacterium glutamicum ATCC13032 and Brevibacterium flavum JV16 were engineered for l-valine production by over-expressing ilvEBN ^r C genes at 31?°C in 72?h fermentation. Different strategies were carried out to reduce the by-products’ accumulation in l-valine fermentation and also to increase the availability of precursor for l-valine biosynthesis. The native promoter of ilvA of C. glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of l-isoleucine. Effect of different relative dissolved oxygen on l-valine production and by-products’ formation was recorded, indicating that 15?% saturation may be the most appropriate relative dissolved oxygen for l-valine fermentation with almost no l-lactic acid and l-glutamate formed. To minimize l-alanine accumulation, alaT and/or avtA was inactivated in C. glutamicum and B. flavum, respectively. Compared to high concentration of l-alanine accumulated by alaT inactivated strains harboring ilvEBN ^r C genes, l-alanine concentration was reduced to 0.18?g/L by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ^r C, and 0.22?g/L by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ^r C. Meanwhile, l-valine production and conversion efficiency were enhanced to 31.15?g/L and 0.173?g/g by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ^r C, 38.82?g/L and 0.252?g/g by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ^r C. This study provides combined strategies to improve l-valine yield by minimization of by-products’ production.     

Association of plasma ortho-tyrosine/para-tyrosine ratio with responsiveness of erythropoiesis-stimulating agent in dialyzed patients

Abstract

Objectives

Patients with end-stage renal failure (ESRF) treated with erythropoiesis-stimulating agents (ESAs) are often ESA-hyporesponsive associated with free radical production. Hydroxyl free radical converts phenylalanine into ortho-tyrosine, while physiological isomer para-tyrosine is formed enzymatically, mainly in the kidney. Production of ‘para-tyrosine’ is decreased in ESRF and it can be replaced by ortho-tyrosine in proteins. Our aim was to study the role of tyrosines in ESA-responsiveness.

Methods

Four groups of volunteers were involved in our cross-sectional study: healthy volunteers (CONTR; n = 16), patients on hemodialysis without ESA-treatment (non-ESA-HD; n = 8), hemodialyzed patients with ESA-treatment (ESA-HD; n = 40), and patients on continuous peritoneal dialysis (CAPD; n = 21). Plasma ortho-, para-tyrosine, and phenylalanine levels were detected using a high performance liquid chromatography (HPLC)-method. ESA-demand was expressed by ESA-dose, ESA-dose/body weight, and erythropoietin resistance index₁ (ERI₁, weekly ESA-dose/body weight/hemoglobin).

Results

We found significantly lower para-tyrosine levels in all groups of dialyzed patients when compared with control subjects, while in contrast ortho-tyrosine levels and ortho-tyrosine/para-tyrosine ratio were comparatively significantly higher in dialyzed patients. Among groups of dialyzed patients the ortho-tyrosine level and ortho-tyrosine/para-tyrosine ratio were significantly higher in ESA-HD than in the non-ESA-HD and CAPD groups. There was a correlation between weekly ESA-dose/body weight, ERI₁, and ortho-tyrosine/para-tyrosine ratio (r = 0.441, P = 0.001; r = 0.434, P = 0.001, respectively). Our most important finding was that the ortho-tyrosine/para-tyrosine ratio proved to be an independent predictor of ERI₁ (β = 0.330, P = 0.016). In these multivariate regression models most of the known predictors of ESA-hyporesponsiveness were included.

Discussion

Our findings may suggest that elevation of the ratio of ortho-tyrosine/para-tyrosine could be responsible for decreased ESA-responsiveness in dialyzed patients.     

A novel bioluminogenic assay for α-chymotrypsin

The use of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin as a novel substrate for α-chymotrypsin has been demonstrated. The kinetic parameters determined are K_M = 0.38mmol/L, k_cat = 6.5 s^?1 and k_cat/k_M = 17,100 (L/mols). The test principle of the coupled assay is the release of aminoluciferin by enzymatic cleavage of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin. Aminoluciferin is oxidized, with light emission, by firefly luciferase (Photinus pyralis) and can be quantified in a luminometric assay. The detection limit for chymotrypsin was found to be 0.3 ng per assay. 6-(N-acetyl-L -phenylalanyl)-aminoluciferin has been synthesized as an example for a new class of highly sensitive substrates. By modification of the peptide residue these new substrates may be suitable for ultrasensitive detection of different proteinases.     

1H- and 13C-NMR studies of N-acetyl-L-alanine methylester and N-acetyl-L-alanine methylamide. I. Self-association

The ¹H- and ¹³C-NMR spectra of N-acetyl-L -alanine methylester and N-acetyl-L -alanine methylamide were measured to examine the modes of self-association of these molecules in solution. The different dilution shifts between these molecules seem to correspond to the difference in the associated state for each molecule. Consequently, for the former molecule, a dimer model forming the intermolecular hydrogen bond through Ala NH hydrogen atom in one molecule to Ala C?O oxygen atom in another molecule was proposed. Another dimer model, which coincides with that proposed recently by Neel and coworkers, was proposed for the latter molecule. This second dimer model forms an intermolecular hydrogen bond through the NH of the N-methylamide group in one molecule to the acetyl C?O in another molecule.