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PubMed2006

Analyses of (1-chloroethenyl)oxirane headspace and hemoglobin N-valine adducts in erythrocytes indicate selective detoxification of (1-chloroethenyl)oxirane enantiomers.

Chloroprene (2-chloro-1,3-butadiene, CAS 126-99-8, CP) is a colorless volatile liquid used in manufacture of polychloroprene, a synthetic rubber polymer. National Toxicology Program inhalation studies of CP in rats and mice gave clear evidence of carcinogenic activity. CP is metabolized by CYP2E1 to electrophilic epoxides, including R- and S-(1-chloroethenyl)oxirane (CEO), which form adducts with nucleic acids and other nucleophiles including glutathione and hemoglobin. As detection of these epoxide metabolites in vivo is technically challenging, measurements of CEO-Hb adducts may provide biomarkers of exposure to bioactivated metabolites of CP. The present studies involved exposure of C57BL/6 mouse erythrocytes (RBC) in vitro to pure enantiomers of CEO. Headspace analysis of CEO using Cyclodex-B capillary GC/MS with selected ion monitoring enabled separation, specific detection, and quantification of CEO enantiomers as reactions proceeded in vitro with RBC. These analyses indicated that R-CEO was much more persistent when incubated in vitro with RBC, while S-CEO disappeared rapidly. After periods of exposure of RBC to various concentrations of R- or S-CEO, erythrocytes were lysed and globin isolated. Covalent adducts, formed by reaction of CEO with N-terminal valine in Hb, were analyzed following Edman cleavage and trimethylsilylation. SIM-GC/MS analyses using a 5%-phenyl-dimethylsiloxane capillary column enabled quantification of CEO-Hb adducts. These analyses produced two chromatographic peaks of CEO-valine adduct derivatives, which were tentatively identified from mass spectra, reaction, and abundance data to be 1-(3-chloro-2-trimethylsilyloxybut-3-en-1-yl)-5-isopropyl-3-phenyl-2-thiohydantoin and 1-[2-chloro-1-(trimethylsilyloxymethyl)prop-2-en-1-yl]-5-isopropyl-3-phenyl-2-thiohydantoin. Analyses quantified significantly greater levels of adducts formed from R-CEO than from S-CEO. Studies involving pretreatment of RBC with glutathione-depleting diethyl maleate diminished the selective detoxification of S-CEO, and suggest enantiomeric selectivity of mouse glutathione-S-transferase as a mechanism of differential detoxification of CEO enantiomers. These results indicate more rapid detoxification of S-CEO by mouse RBC in vitro, while R-CEO may persist to react with cellular nucleophiles.

Animals↗
PubMed2006

Quantitative analysis of N-terminal valine peptide adducts specific for 1,2-epoxy-3-butene.

Butadiene (BD) metabolism shows gender, species and concentration dependency, making the extrapolation of animal results to humans complex. BD is metabolized mainly by cytochrome P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2;3,4-diepoxybutane (DEB) and 1,2-epoxy-butanediol (EB-diol). For accurate risk assessment it is important to elucidate species differences in the internal formation of the individual epoxides in order to assign the relative risks associated with their different mutagenic potencies. Analysis of N-terminal globin adducts is a common approach for monitoring the internal formation of BD derived epoxides. Our long term strategy is to develop an LC-MS/MS method for simultaneous detection of all three BD hemoglobin adducts. This approach is modeled after the recently reported immunoaffinity LC-MS/MS method for the cyclic N,N-(2,3-dihydroxy-1,4-butadyil)-valine (pyr-Val, derived from DEB). We report herein the analysis of the EB-derived 2-hydroxyl-3-butenyl-valine peptide (HB-Val). The procedure utilizes trypsin hydrolysis of globin and immunoaffinity (IA) purification of alkylated heptapeptides. Quantitation is based on LC-MS/MS monitoring of the transition from the singly charged molecular ion of HB-Val (1-7) to the a(1) fragment. Human HB-Val (1-11) was synthesized and used for antibody production. As internal standard, the labeled rat-[(13)C(5)(15)N]-Val (1-11) was prepared through direct alkylation of the corresponding peptide with EB. Standards were characterized and quantified by LC-MS/MS and LC-UV. The method was validated with different amounts of human HB-Val standard. The recovery was >75% and coefficient of variation <25%. The LOQ was set to 100 fmol/injection. For a proof of principal experiment, globin samples from male and female rats exposed to 1000 ppm BD for 90 days were analyzed. The amounts of HB-Val present were 268.2+/-56 and 350+/-70 pmol/g (mean+/-S.D.) for males and females, respectively. No HB-Val was detected in controls. These data are much lower compared to previously reported values measured by GC-MS/MS. The difference may be due higher specificity of the LC-MS/MS method to the N-terminal peptide from the alpha-chain versus derivatization of both alpha- and beta-chain by Edman degradation, and possible instability of HB-Val adducts during long term storage (about 10 years) between the analyses. These differences will be resolved by examining recently collected samples, using the same internal standard for parallel analysis by GC-MS/MS and LC-MS/MS. Based on our experience with pyr-Val adduct assay we anticipate that this assay will be suitable for evaluation of HB-Val in multiple species.

Animals↗
PubMed2004

Attenuation of paraquat-induced motor behavior and neurochemical disturbances by L-valine in vivo.

Alterations of motor behavioral patterns and monoamine contents in the discrete rat brain areas after acute paraquat exposure (3, 5, 10, 20 mg/kg, s.c.) have been studied. The results showed that paraquat at the doses of 5, 10, and 20 mg/kg significantly reduced locomotive, stereotypic, and rotational behaviors. Significant decreases of norepinephrine (NE) contents in cortex and hypothalamus, as well as striatal contents of dopamine (DA) and its acidic metabolites, were detected. In addition, L-valine (200 mg/kg, i.p.) significantly attenuated paraquat-induced toxicity at moderate dose (5 mg/kg) but not at high dose (20 mg/kg). The results provide evidence that paraquat can enter the brain as illustrated by the alterations in the motor behavioral pattern and neurochemical contents. Furthermore, the attenuation effect of L-valine against systemic administration of paraquat-induced motor behaviors was detected, with a slightly protective effect on paraquat-induced neurochemical alterations.

Animals↗
PubMed2003

Enantiomeric separation using temperature-responsive chiral polymers composed of L-valine diamide derivatives in aqueous liquid chromatography.

This paper describes enantiomer separation by aqueous liquid chromatography using chiral stationary phases (CSPs) in which temperature-responsive polymers derived from acryloyl-L-valine N-methylamide (1) and its N,N-dimethylamide analogue (2) were bound on silica gel supports. The linear polymers composed of monomer 1 and monomer 2 are temperature-responsive in solution and their aggregation and extension states related to water solubility are reversible at particular critical temperatures. During chromatography, enantioselectivity and retentivity for solute enantiomers were controlled by column temperature, which changes the aggregation and extension states of the chiral polymers depending upon their interior hydrophobic nature. Two different types of CSPs were made: a temperature-responsive linear polymer derived from 3-mercaptopropyl silica gel, and another polymer cross-linked with ethylene dimethacrylate from 3-methacryloyloxypropyl silica gel. The former CSP could separate racemic N-(3,5-dinitrobenzoyl(DNB))amino acid isopropyl esters. Retention of the amino acid derivatives was prolonged with an increase in column temperature. Enantioselectivity was also enhanced with temperature increase until the particular critical temperature. The latter, cross-linked CSP could not provide enantioselectivity for the amino acid derivatives in aqueous media, although the chiral valine diamide moieties were effective for enantiomer separation in non-aqueous media. The degree of hydrophobicity and volume of the bonded phase formed by the polymers on the support surface was determined by measuring the fluorescence of pyrene.

Chromatography, Liquid↗
PubMed1985

Biochemical changes during valine production by Aerobacter aerogenes.

The typical chemical changes taking place during valine production by A. aerogenes in a synthetic medium have been investigated. The principal carbon and nitrogen sources, sucrose and urea, respectively, have been found to be almost exhausted during an incubation period lasting 90 h. While the carbon compound was depleted right from the start of formentation, the disappearance of urea was noticeable after 16 h of incubation. The formation of valine in the broth started between 24 and 40 h of incubation connected with the rapid utilization of urea. The pH value of the broth was found to glide down from an initial value of 7.5 to 6.4 within 24 h then rising again to 7.5 during 64 h and further to 8.0 in 90 h of incubation.

Culture Media↗
PubMed2000

Protease inhibitors. Part 7. Inhibition of Clostridium histolyticum collagenase with sulfonylated derivatives of L-valine hydroxamate.

Sulfonylated L-valine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with the title amino acid, followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group. Other derivatives were obtained by reaction of N-benzyl-L-valine with arylisocyanates, arylsulfonylisocyanates or benzoylisothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety, with hydroxylamine in the presence of carbodiimides. The obtained compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical collagen. The hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized derivatives, substitution patterns leading to best ChC inhibitors were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-; 3-trifluoromethylphenylsulfonyl; or 1- and 2-naphthyl among others. Similarly to the matrix metalloproteinase hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P(2') and P(3') subsites, in order to achieve tight binding to the enzyme. Such compounds might lead to drugs useful in the treatment corneal bacterial keratitis.

Hydroxamic Acids↗
PubMed1998

Mutation of aspartate-233 to valine in mouse ornithine decarboxylase reduces enzyme activity.

Ornithine decarboxylase is the first and key enzyme in mammalian polyamine biosynthesis. All eukaryotic ornithine decarboxylases contain several highly conserved regions and the amino acid residues 232-238 form one of the most highly conserved sequences. This region contains a glycine-rich sequence typically found in a number of pyridoxal 5'-phosphate-dependent or nucleotide-binding proteins. We mutated aspartate-233 which is the only acidic residue within this region to valine. This mutation causes striking sequence similarity with the guanine nucleotide binding domain of c-H-ras. Mutated ornithine decarboxylase cDNA with a mouse mammary tumor virus long terminal repeat promoter has been transfected for stable expression into ornithine decarboxylase-deficient C55.7 cells. Ornithine decarboxylase activity of the mutated enzyme was about 20% of wild-type ornithine decarboxylase activity and it was not activated by guanosine triphosphate like the ornithine decarboxylase isoform found in some tumors and rat brain. The mutation caused an increase in K(m) value of about 20-fold both for the substrate L-ornithine and for the cofactor pyridoxal 5'-phosphate. The Ki value for the irreversible inhibitor alpha-difluoromethylornithine was also increased, whereas the half-life of the enzyme was shortened. These results suggest that the region containing aspartate-233 is essential for binding of the cofactor and thus forms part of enzymatic active site, and the mutation of aspartate-233 to valine cannot, at least alone, cause the activation of ornithine decarboxylase by guanosine triphosphate (230).

Amino Acid Sequence↗
PubMed2003

Diagnosis of maple syrup urine disease by determination of L-valine, L-isoleucine, L-leucine and L-phenylalanine in neonatal blood spots by gas chromatography-mass spectrometry.

A novel method was developed for the diagnosis of maple syrup urine disease (MSUD) by the determination of L-valine, L-leucine, L-isoleucine and L-phenylalanine in dried blood spots of newborns by gas chromatography-mass spectrometry (GC-MS). The four amino acids were extracted from blood samples by methanol and derivatized by n-butanol and trifluroacetic anhydride under optimum reaction conditions. The corresponding single derivatives of the four amino acids were obtained under the optimum conditions. Their contents in blood samples were analyzed quantitatively by measurement of their derivatives by GC-MS in selected ion monitoring mode. MSUD can be diagnosed on the basis of the ratio of the total content of L-leucine and L-isoleucine to that of L-phenylalanine. The present method only took a short time to perform and required minimal sample preparation, which provided low detection limits and a relative standard deviation of less than 5.0%. The derivatization reactions of the four amino acids, L-valine, L-isoleucine, L-leucine and L-phenylalanine, with n-butanol and trifluroacetic anhydride were investigated and the optimum reaction conditions, including reaction time and temperature, were obtained and used for the determination of the amino acids in plasma samples.

Gas Chromatography-Mass Spectrometry↗
PubMed1982

Effect of a valine load test on plasma alpha-keto acids in Friedreich ataxia.

To test the physiological significance in vivo of our previous in vitro finding of reduced valine dehydrogenase (VDH) activity in patients with Friedreich's Ataxia, we subjected ataxic patients and controls to an oral valine load test (1.0g) and measured the levels of branched chain alpha-keto acids in the plasma for 24 hours. We demonstrated a significantly higher peak for a alpha-keto isovaleric acid in Friedreich's Ataxia and a general trend towards higher than control values in all other alpha-keto acids measured, and at all times in the experiment. These changes are compatible with the postulated defect in regulation of the activity of VDH in this illness, but because of their small amplitude, they also indicate that a VDH deficiency is not the genetic defect in Friedreich's Ataxia.

Adult↗
PubMed2006

Combining the enantioselectivities of L-valine diamide and permethylated beta-cyclodextrin in one gas chromatographic chiral stationary phase.

An L-valine diamide chiral selector was attached to a polysiloxane through a long hydrocarbon spacer giving rise to a chiral stationary phase (CSP), Chirasil-Val-C11. The enantioselective properties of this readily accessible diamide CSP under gas chromatographic conditions were found to be similar to that of the commercially available Chirasil-Val CSP prepared by a polymer-analogous route. A new binary CSP, Chirasil-DexVal-C11, was synthesized by means of simultaneous attachment of both the L-valine diamide and permethylated beta-cyclodextrin selectors to a polysiloxane using platinum-catalyzed hydrosilylation, thereby overcoming the immiscibility problem known for Chirasil-Val and Chirasil-Dex. This binary CSP retained both the enantioselectivity of Chirasil-Val-C11 toward alpha-amino acid derivatives and the unsurpassed enantioselectivity of Chirasil-Dex toward underivatized chiral alcohols, ketones, and hydrocarbons. Furthermore, it was shown that the presence of the cyclodextrin selector in Chirasil-Val-C11 significantly improved the enantioseparation of proline, which represented a problematic amino acid on diamide CSPs.

Alcohols↗
PubMed1981

In vitro conversion of leucine to valine: configurational assignment of [5-13C]leucines.

Chiral (2S)-[5-13C]leucine was obtained from Escherichia coli deficient in the synthesis of acetolactate when cultures were supplemented with (RS)-[2-13CH3]acetolactate. The carbon-13 nuclear magnetic resonance spectrum showed one strong peak with a chemical shift of 21.4 ppm relative to tetramethylsilane [Sylvester, S. R., & Stevens, C. M. (1979) Biochemistry 18, 4529-4531]. Silver picolinate oxidation of the labeled leucine gave isovaleric acid which was then brominated at the alpha position to give (2RS)-2-bromo[3-13CH3]-isovaleric acid (2-bromo-3-[13C]methylbutanoic acid). Aminolysis afforded (2RS)-[4-13C]valine which was treated with D-amino acid oxidase in the presence of catalase. The final product was identified as (2S,3S)-[4-13C]valine by the specificity of D-amino acid oxidase, by amino acid analysis, and by the persistence of a strong signal at gamma 17.8 in the carbon-13 magnetic resonance spectrum. These results establish the absolute configuration of the biosynthetic leucine to be (2S,4S)-[5-13C]leucine.

Carbon Isotopes↗
PubMed1976

Chromatic models. Interactions between DNA and polypeptides containing L-lysine L-valine: circular dichroism and thermal denaturation studies.

The interaction of calf thymus DNA with statistical copolymers of L-lysine and L-valine [poly(L-Lys100f-Lvalf)] and block copolymers [poly(L-Lys)100f-poly(L-Val)f] were investigated as a function of ionic strength using circular dichroism (CD) spectroscopy. It was found that valine suppresses the ability of the copolymer-DNA complexes to yield a psi-type CD spectra as found for poly(L-Lys)-DNA [Jordan, C.F., Lerman, L.S., and Venable, J.N. (1972), Nature (london), New Biol. 236, 67] and lowers the ionic strength at which CD distortion occurs. Thermal denaturation, simultaneously monitoring 280-nm ellipticity, [theta]280, and hyperchromicity, h280, was carried out on annealed complexes of poly(L-Lys)-DNA, poly(L-Lys84.5-L-Val15.5)-DNA, poly(L-Lys)87.2-poly(L-Val)12.8-DNA, and directly mixed complexes of poly(L-Lys)-DNA, IN 2.5 X 10(-4) MEDTA, pH 7.0 solution. The CD denaturation of uncomplexed DNA at several ionic strengths was also determined to examine pre-melting. Despite the inability of both statistical and block copolymers of L-Lys and L-Val to form psi-type complexes with DNA, they bind as well to DNA as does poly(L-Lys) and give rise to a thermal denaturation pattern showing bound peaks between 90 and 100 degrees C, seen clearly with CD denaturation. The thermal denaturation of mixed and annealed complexes of poly(L-Lys)-DNA shows similar patterns in hyperchromicity changes as a function of temperature but very different CD melts. From the CD melt of annealed poly(L-Lys)-DNA, it appears that aggregation and long-range order of the complex are significant in low salt (2.5 X 10(-4) MEDTA) as well as in 1.0 M NaCl. These studies further illustrate the importance of the nature of nonionic interactions (hydrophobic) between polypeptides and DNA in determining the behavior of their complexes, such as causing condensation into higher order asymmetric structures. In light of these observations, the possible significance to the CD melting of chromatin and the validity of identification of C-form DNA by CD spectroscopy are discussed.

Chromatin↗
PubMed2004

Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation.

A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. We have employed purified and reconstituted P-glycoproteins expressed in Saccharomyces cerevisiae [Figler et al. (2000) Arch. Biochem. Biophys. 376, 34-46] and devised a set of thermodynamic analyses to reveal the mechanism of improved resistance. Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicine- and etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. The mutant enzyme has a higher apparent K(m) for colchicine and a lower K(m) for etoposide than that of wild type. Kinetic constants for other transported drugs were not significantly modified by this mutation. Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicine- and etoposide-dependent activities. To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. The high transition state energies of wild-type P-glycoprotein, when transporting etoposide or colchicine, increase the probability of nonproductive degradation of the transition state without transport. G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Our new data fully reconcile the apparently conflicting results of previous studies. EPR analysis of the spin-labeled G185C enzyme in a cysteine-less background and kinetic parameters of the G185C enzyme indicate that position 185 is surrounded by other residues and is volume sensitive. These results and atomic detail structural modeling suggest that residue 185 is a pivotal point in transmitting conformational changes between the catalytic sites and the colchicine drug binding domain. Replacement of this residue with a bulky valine alters this communication and results in more efficient transport of etoposide or colchicine.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Synthetase recognition determinants of E. coli valine transfer RNA.

We have studied the interactions between Escherichia coli tRNAVal and valyl-tRNA synthetase (ValRS) by enzymatic footprinting with nuclease S1 and ribonuclease V1, and by analysis of the aminoacylation kinetics of mutant tRNAVal transcripts. Valyl-tRNA synthetase specifically protects the anticodon loop, the 3' side of the stacked T-stem/acceptor-stem helix, and the 5' side of the anticodon stem of tRNAVal against cleavage by double- and single-strand-specific nucleases. Increased nuclease susceptibility at the ends of the anticodon- and T-stems in the tRNAVal.ValRS complex is indicative of enzyme-induced conformational changes in the tRNA. The most important synthetase recognition determinants are the middle and 3' anticodon nucleotides (A35 and C36, respectively); G20, in the variable pocket, and G45, in the tRNA central core, are minor recognition elements. The discriminator base, position 73, and the anticodon stem also are recognized by ValRS. Replacing wild-type A73 with G73 reduces the aminoacylation efficiency more than 40-fold. However, the C73 and U73 mutants remain good substrates for ValRS, suggesting that guanosine at position 73 acts as a negative determinant. The amino acid acceptor arm of tRNAVal contains no other synthetase recognition nucleotides, but regular A-type RNA helix geometry in the acceptor stem is essential [Liu, M., et al. (1997) Nucleic Acids Res. 25, 4883-4890]. In the anticodon stem, converting the U29:A41 base pair to C29:G41 reduces the aminoacylation efficiency 50-fold. This is apparently due to the rigidity of the anticodon stem caused by the presence of five consecutive C:G base pairs, since the A29:U41 mutant is readily aminoacylated. Identity switch experiments provide additional evidence for a role of the anticodon stem in synthetase recognition. The valine recognition determinants, A35, C36, A73, G20, G45, and a regular A-RNA acceptor helix are insufficient to transform E. coli tRNAPhe into an effective valine acceptor. Replacing the anticodon stem of tRNAPhe with that of tRNAVal, however, converts the tRNA into a good substrate for ValRS. These experiments confirm G45 as a minor ValRS recognition element.

Anticodon↗
PubMed2000

Valine of the YVDD motif of moloney murine leukemia virus reverse transcriptase: role in the fidelity of DNA synthesis.

The YXDD motif is highly conserved in the reverse transcriptase family. The variable X residue is occupied by valine and methionine in MuLV RT and HIV-1 RT, respectively. Previous studies have shown that Tyr 222, the Y residue of the YXDD motif in MuLV RT, constitutes a major component of the fidelity center of the enzyme [Kaushik, N., Singh, K., Alluru, I., and Modak, M. J. (1999) Biochemistry 38, 2617-2627]. In this work, we present evidence that reverse transcriptases containing valine in the "X" position of the YXDD motif generally catalyze DNA synthesis with greater fidelity than those containing methionine or alanine. In the MuLV RT system, the two mutants V223M and V223A exhibited an overall reduced fidelity of DNA synthesis, specifically for RNA-templated reactions. Further analysis revealed that these mutants exhibit a higher efficiency of misinsertion on MS2 RNA than the wild-type enzyme for every mispair tested. However, unlike HIV-1 RT, the insensitivity of the wild-type MuLV RT to all four ddNTPs remained unchanged by mutation of V223 to Met or Ala. A 3D molecular model of the ternary complex of MuLV RT, template primer, and dNTP suggests that Val 223 along with its neighboring Tyr 222 stabilizes the substrate binding pocket via hydrophobic interactions with the dNTP substrate and template-primer.

Animals↗
PubMed2003

Binding energies of water to sodiated valine and structural isomers in the gas phase: the effect of proton affinity on zwitterion stability.

The structures of valine (Val) and methylaminoisobutyric acid (Maiba) bound to a sodium ion, both with and without a water molecule, are investigated using both theory and experiment. Calculations indicate that, without water, sodiated Val forms a charge-solvated structure in which the sodium ion coordinates to the nitrogen and the carbonyl oxygen (NO-coordination), whereas Maiba forms a salt-bridge structure in which the sodium ion coordinates to both carboxylate oxygens (OO-coordination). The addition of a single water molecule does not significantly affect the relative energies or structures of the charge-solvated and salt-bridge forms of either cluster, although in Maiba the mode of sodium ion binding is changed slightly by the water molecule. The preference of Maiba to adopt a zwitterionic form in these complexes is consistent with its higher proton affinity. Experimentally, the rates of water evaporation from clusters of Val.Na(+)(H(2)O) and Maiba.Na(+)(H(2)O) are measured using blackbody infrared radiative dissociation (BIRD). The dissociation rates from the Val and Maiba complexes are compared to water evaporation rates from model complexes of known structure over a wide range of temperatures. Master equation modeling of the BIRD kinetic data yields a threshold dissociation energy for the loss of water from sodiated valine of 15.9 +/- 0.2 kcal/mol and an energy of 15.1 +/- 0.3 kcal/mol for the loss of water from sodiated Maiba. The threshold dissociation energy of water for Val.Na(+)(H(2)O) is the same as that for the charge-solvated model isomers, while the salt-bridge model complex has the same water threshold dissociation energy as Maiba.Na(+)(H(2)O). These results indicate that the threshold dissociation energy for loss of a water molecule from these salt-bridge complexes is approximately 1 kcal/mol less than that for loss of water from the charge-solvated complexes.

Butyrates↗
PubMed2001

N-methylcarbamoylated valine of hemoglobin in humans after exposure to N,N-dimethylformamide: evidence for the formation of methyl isocyanate?

N,N-Dimethylformamide (DMF) is reported to cause testicular germ-cell tumors in exposed workers. The reports, however, are not in line with results obtained in animal and in vitro experiments, where DMF was shown not to be mutagenic and also not to be carcinogenic. Considerable interest raised on the formation of a reactive intermediate, presumably methyl isocyanate (MIC), during metabolism of DMF in humans over the last years. We report the formation of N-methylcarbamoylated valine of hemoglobin (Hb) in blood samples from workers exposed to DMF in the polyacrylic fiber industry. N-Methylcarbamoylated Hb was formed by the reaction of MIC with Hb. For this purpose, Hb adducts were monitored by means of a modified Edman degradation involving the release of the N-terminal valine adduct in form of 3-methyl-5-isopropylhydantoin (MIH). For internal standardization of the method, 3-ethyl-5-isopropylhydantoin (EIH) was used. Separation and analysis of MIH and EIH were carried out by gas chromatography and mass spectrometry with electron impact ionization (GC/EI-MS). Hb adducts in form of MIH were quantified in blood samples from exposed persons in concentrations between 26.1 and 412.0 nmol of MIH/g of globin. The observed adducts were proven to be identical to those derived from the in situ reaction between Hb and MIC. Taken together with the fact that only N-methylcarbamoylated Hb can undergo ring closure to the corresponding hydantoin, the reaction is indirect evidence for the occurrence of MIC in vivo. The formation of MIC directly in the cell and its possible distribution through the human body may lead to critical effects after exposure to DMF. Adducts were determined not to be totally specific for exposure to DMF since an identical adduct was also found in blood samples from the general population. However, concentrations were lower by a factor of about 100. The sources for background adducts are currently unknown.

Antisickling Agents↗
PubMed2000

Alteration of substrate specificity of valine dehydrogenase from Streptomyces albus.

The catabolism of branched chain amino acids, especially valine, appears to play an important role in furnishing building blocks for macrolide and polyether antibiotic biosyntheses. To determine the active site residues of ValDH, we previously cloned, partially characterized, and identified the active site (lysine) of Streptomyces albus ValDH. Here we report further characterization of S. albus ValDH. The molecular weight of S. albus ValDH was determined to be 38 kDa by SDS-PAGE and 67 kDa by gel filtration chromatography indicating that the enzyme is composed of two identical subunits. Optimal pHs were 10.5 and 8.0 for dehydrogenase activity with valine and for reductive amination activity with alpha-ketoisovaleric acid, respectively. Several chemical reagents, which modify amino-acid side chains, inhibited the enzyme activity. To examine the role played by the residue for enzyme specificity, we constructed mutant ValDH by substituting alanine for glycine at position 124 by site-directed mutagenesis. This residue was chosen because it has been considered to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). The Ala-124-Gly mutant enzyme displayed lower activities toward aliphatic amino acids, but higher activities toward L-phenylalanine, L-tyrosine, and L-methionine compared to the wild type enzyme suggesting that Ala-124 is involved in substrate binding in S. albus ValDH.

Amino Acid Oxidoreductases↗