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PubMed2000

Mutation of the surface valine residues 8 and 44 in the rubredoxin from Clostridium pasteurianum: solvent access versus structural changes as determinants of reversible potential.

The Pr(i) sidechains of two adjacent valine residues, V8 and V44, define the surface of the rubredoxin from Clostridium pasteurianum and control access to its Fe(S-Cys)4 active site. To assess the effect of systematic change of the steric bulk of the alkyl sidechains, eight single and three double mutant proteins have been isolated which vary G (H), A (Me), V (Pr(i)), L (Bu(i)) and I (Bu(s)) at those positions. X-ray crystal structures of the Fe(III) forms of the V44A and V44I proteins are reported. Positive shifts in reversible potential of up to 116 mV are observed and attributed to increased polarity around the Fe(S-Cys)4 site induced by (1) changes in protein backbone conformation driven by variation of the steric demands of the sidechain substituents and (2) changes in solvent access to the side-chains of ligands C9 and C42. Data for the V44A mutant show that a minor change in the steric requirements of a surface residue can introduce a NH...Sgamma hydrogen bond at the active site and lead to a shift in potential of + 50 mV.

Base Sequence↗
PubMed1998

N-methylcarbamoyl adducts at the N-terminal valine of globin in workers exposed to N,N-dimethylformamide.

N,N-dimethylformamide (DMF) is a commonly used industrial solvent. The formation of some metabolites of DMF in humans occurs via N-methyl-carbamoylated species (e.g. N-methylcarbamoylated glutathione). The aim of our study was to investigate whether DMF leads to N-methylcarbamoylated adducts at the N-terminal valine of haemoglobin (Hb). Therefore, Hb adduct levels of ten DMF exposed workers and ten controls were analysed by a specific and sensitive detection method using capillary gas chromatography and a mass selective detector (GC/MS). Using this method we were able to show for the first time that Hb adducts are formed during the metabolism of DMF in humans. The general population, however, shows still unidentified background levels of this adduct which are on average lower by a factor of 50. The pathway for the formation of the investigated DMF-Hb adduct in workers exposed to DMF is still unknown. As identical adducts were also found after exposure to methylisocyanate (MIC), our work indicates the formation of MIC during the metabolism of DMF. The formation of Hb adducts with DMF and its relevance for occupational health is a subject of further research.

Acetylcysteine↗
PubMed2005

Accurate determination of leucine and valine side-chain conformations using U-[15N/13C/2H]/[1H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cgamma-Hgamma/Cbeta-Hbeta residual dipolar couplings: application to the 34-kDa enzyme IIA(chitobiose).

An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a 15N/13C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hgamma(i)-H(N)(i) and Hgamma(i)-H(N)(i+1) NOEs in a 3D 15N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine 13C-1H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIA(Chitobiose), a 34 kDa homotrimeric phosphotransferase protein.

Carbon Isotopes↗
PubMed2005

The mitochondrial branched-chain aminotransferase (AtBCAT-1) is capable to initiate degradation of leucine, isoleucine and valine in almost all tissues in Arabidopsis thaliana.

Plants are capable to de novo synthesize the essential amino acids leucine, isoleucine and valine. Studies in recent years, however, also revealed that plants have the potential to degrade leucine or may be all of the branched-chain amino acids. One of the enzymes participating in both biosynthesis and degradation is the branched-chain aminotransferase, which is in Arabidopsis thaliana encoded by a small gene family with six transcribed members. We have now studied the steady state mRNA levels by quantitative RT-PCR and promoter activities of these genes with promoter::glucuronidase reporter gene constructs in transgenic plants. The gene encoding the mitochondrial isoenzyme (Atbcat-1) is expressed in all tissues with predominant transcription in seedlings and leaves. Surprisingly the plastid located proteins (AtBCAT-2, -3 and -5) are expressed at rather low levels with only Atbcat-3 transcribed in all tissues. The most likely cytoplasmic-located AtBCAT-4 and AtBCAT-6 are mainly expressed in tissues associated with transport function and in meristematic tissues, respectively. A detailed characterization of the enzyme activity and substrate specificity of the mitochondrial AtBCAT-1 enzyme revealed the potential of this enzyme to initiate degradation of all branched-chain amino acids. In addition alpha-aminobutyrate and alpha-ketobutyrate as well as methionine and alpha-ketomethylthiobutyrate are identified as substrates. This suggests that AtBCAT-1 and potentially other members of this protein family may influence methionine levels and may play an important role in the metabolism of the nonprotein amino acid alpha-aminobutyrate. The consequences of these substrate specificities for bioplastic production and methionine homeostasis are discussed.

Amino Acids, Branched-Chain↗
PubMed1992

Ring test for low levels of N-(2-hydroxyethyl)valine in human hemoglobin.

Hemoglobin adducts are useful for the identification and quantification of electrophilic agents in vivo. A modified Edman degradation method has been extensively used for monitoring exposure to ethylene oxide through gas chromatographic-mass spectrometric measurements of hydroxyethyl adducts to the N-terminal valines in hemoglobin. In a ring test, four laboratories using different versions of the method analyzed eight human globin samples with low adduct levels from ethylene oxide. Measurements of the same adduct by a radioimmunoassay were also included. Strong correlation between the measurements by the different laboratories shows that the method in principle works well. However, there were some systematic quantitative differences.

Hemoglobins↗
PubMed1975

Effect of modification on physicochemical and biological properties of haptoglobin. VI. Reaction with azlactone of p-nitrobenzoyl-valine.

The azlactone of p-nitrobenzoyl-valine (Nbz-Val) has been used for modification of xi-amino groups of lysine in haptoglobin type 1-1, in hemoglobin, and in the haptoglobin-hemoglobin complex. By the use of this reagent 95% of amino groups in haptoglobin and 90% in hemoglobin have been blocked without any changes in peroxidase activity of the formed complexes: Nbz-Val.haptoglobin with hemoglobin, Nbz-Val. hemoglobin with haptoglobin, and Nbz-Val.(haptoglonin-hemoglobin). After reduction and reoxidation, Nbz-Val.haptoglobin was found to retain 90% of peroxidase activity when complexed with hemoglobin. Beta chains separated either from haptoglobin or Nbz-Val.haptoglobin showed 15% of peroxidase activity in the complex with hemoglobin, alpha chains of the same origin were completely inactive. Whereas recombination of haptoglobin from alpha and beta chains resulted in 42% hemoglobin-binding capacity, renaturation of Nbz-Val.haptoglobin from separated subunits was found to proceed with almost 100% yield. In immunodiffusion with rabbit anti-haptoglobin or anti-Nbz-Val.haptoglobin sera, preparations of haptoglobin and Nbz-Val.haptoglobin after reduction and reoxidation or after recombination from separated subunits gave similar precipitation arcs showing the reaction of immunological identity.

Haptoglobins↗
PubMed1994

Detection of 4'-phosphopantetheine at the thioester binding site for L-valine of gramicidinS synthetase 2.

Biosynthesis of gramicidinS in Bacillus brevis is catalysed by a multienzyme system consisting of two multifunctional proteins, gramicidinS synthetase 1 and 2 codified by the grsA and grsB genes, respectively. GramicidinS synthetase 2 shows a modular architecture of four amino acid-activating domains each containing a thioester binding motif LGG H/D S L/I highly conserved in its C-terminal region, as demonstrated by sequence analysis of the grsB gene [W. Schlumbohm et al. (1991) J. Biol. Chem. 266, 23135-23141]. This multienzyme was specifically labeled at the thioester binding site of L-valine with [3H]N-ethylmaleimide using a substrate protection technique. After enzymatic digestion a labeled active site peptide was isolated in pure form by multistep methodology. This fragment was identified by gas-phase sequencing as the active site peptide of the thiotemplate site for L-Val by comparison with the grsB gene sequence. By mass spectrometry in combination with amino acid analysis it was demonstrated that a 4'-phosphopantetheine carrier was attached to the active serine in this motif. Our results give evidence that multiple peripheral 4'-phosphopantetheine carriers are involved in the formation of gramicidinS in contrast to a central carrier arm as assumed in the original version of the thiotemplate mechanism. A 'Multiple Carrier Model' of nonribosomal peptide biosynthesis is proposed.

Amino Acid Isomerases↗
PubMed1991

In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core.

The crystal structure of the staphylococcal nuclease mutant V66K, in which valine 66 is replaced by lysine, has been solved at 1.97 A resolution. Unlike lysine residues in previously reported protein structures, this residue appears to bury its side-chain in the hydrophobic core without salt bridging, hydrogen bonding or other forms of electrostatic stabilization. Solution studies of the free energy of denaturation, delta GH2O, show marked pH dependence and clearly indicate that the lysine residue must be deprotonated in the folded state. V66K is highly unstable at neutral pH but only modestly less stable than the wild-type protein at high pH. The pH dependence of stability for V66K, in combination with similar measurements for the wild-type protein, allowed determination of the pKa values of the lysine in both the denatured and native forms. The epsilon-amine of this residue has a pKa value in the denatured state of 10.2, but in the native state it must be 6.4 or lower. The epsilon-amine is thus deprotonated in the folded molecule. These values enabled an estimation of the epsilon-amine's relative change in free energy of solvation between solvent and the protein interior at 5.1 kcal/mol or greater. This implies that the value of the dielectric constant of the protein interior must be less than 12.8. Lysine is usually found with the methylene groups of its side-chain partly buried but is nevertheless considered a hydrophilic surface residue. It would appear that the high pKa value of lysine, which gives it a positive charge at physiological pH, is the primary reason for its almost exclusive confinement to the surface proteins. When deprotonated, this amino acid type can be fully incorporated into the hydrophobic core.

Computer Simulation↗
PubMed1980

Influence of recC and uvrD on ultraviolet-induced mutation to valine resistance in E. coli K12.

The production of mutants in E. coli exposed to ultraviolet light is initiated by photochemical reactions, and completed by metabolic processes controlled by recA and other genes. Ultraviolet-induced mutagenesis to valine resistance was measured in cells carrying recC, uvrD, or both recC and uvrD. The spontaneous and UV-induced mutagenesis was slightly greater in those carrying uvrD, as compared to recC or wild-type. At low doses, UV mutagenesis in the recC uvrD double mutant was greater than in either recC or wild-type, and was comparable to that in the uvrD strain, although this double mutant was very UV-sensitive and showed poor survival at doses above 2 J/m2.

Dose-Response Relationship, Radiation↗
PubMed1994

Effects of valine, leucine, isoleucine, and a balanced amino acid solution on the seizure threshold to picrotoxin in rats.

During infusion of branched-chain amino acids (BCAAs) in humans, changes in ventilatory drive, sleeping pattern, and appetite have been reported. The mechanism by which BCAA exerts their effects on CNS remains unclear. An infusion of a BCAA solution (300 mg/kg) has previously been found to increase the seizure threshold in rats to the proconvulsant drug picrotoxin, an antagonist on the GABA-benzodiazepine receptor complex. In this study, each of the BCAAs given separately (valine, leucine, isoleucine; 300 mg/kg) (n = 10) increased the mean latency time to onset of seizures vs. placebo as an indication of an increased seizure threshold. A balanced amino acid solution (Vamin-Glucose) had no effect on the seizure threshold. Thus, these CNS effects are specific for BCAAs and occur with all three.

Amino Acids↗
PubMed1979

Assembly of the mitochondrial membrane system: sequences of yeast mitochondrial valine and an unusual threonine tRNA gene.

The mitochondrial DNA segments of two independently isolated rho- clones of S. cerevisiae carrying a genetic marker for a threonine tRNA have been characterized by restriction endonuclease analysis and DNA sequencing. The DNA sequences of the two segments have been used to deduce the primary and secondary structures of the tRNA. The threonine tRNA is unusual in having a leucine anticodon (3'-GAU-5'). Despite the anomalous anticodon, the tRNA is proposed to function in mitochondrial protein synthesis. One of the rho- clones contains an additional coding sequence that has been identified as a valine tRNA genes have been located on the wild-type physical map and determined to be transcribed from two different strands.

Base Sequence↗
PubMed1989

Interactions of Escherichia coli SO-187 tRNA(IVal) with Bacillus stearothermophilus valine-tRNA synthetase studied by 13C-NMR.

Uracil isotopically labelled with 13C at C4 and C5 has been incorporated into nucleic acids of the Escherichia coli uracil auxotroph, SO-187. [4,5-13C]uracil-labeled tRNA(IVal) was isolated and purified. 13C longitudinal relaxation times measured at 67.8 MHz demonstrated that the C5 dipole caused a 20-50% increase in the C4 relaxation. Interactions of this tRNA with valine-tRNA synthetase (VTS) purified from Bacillus stearothermophilus were established by 13C-NMR. Specific spectral changes were seen at 4-thiouridine, ribothymidine and pseudouridine of the 'bend' in the three-dimensional structure, and particularly at the uridine-5-oxyacetic acid in the wobble position of the anticodon. Thus, the protein seems to be in contact along the entire tRNA molecule, including the anticodon loop.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Comparison of jejunal and ileal absorptive functions for glucose and valine in vivo--a technique for estimating real Km and Jmax in the domestic fowl.

1. A technique is described that enables the kinetic characterisation of the saturable absorption mechanisms in the chicken jejunum and ileum for glucose and valine in vivo (after correction for non-saturating components and unstirred layers) by estimation of real Km and Jmax. 2. In the ileum both nutrients have lower real Km and higher Jmax values than in the jejunum indicating, at least for hexose and amino acids, that the ileal enterocytes are functionally equipped and anatomically well-sited to fulfil the role of scavengers of the small intestine.

Animals↗
PubMed1979

Kinetics and mechanism for the conformational transition in p-guanidinobenzoate bovine trypsinogen induced by the isoleucine-valine dipeptide.

The interaction between p-guanidinobenzoate-trypsinogen and the isoleucine-valine dipeptide has been investigated by temperature-jump relaxation spectrometry. Using the absorbance at 281 nm the concentration dependence of the relaxation parameters is consistent with the conventional induced-fit model: rapid ligand binding coupled to a slower intramolecular change; some alternative mechanisms can be excluded. At 296 K, 0.1 M Tris HCl, pH = 7.4, the dissociation equilibrium constant for the overall process is K = 5.1(+/- 0.2) X 10(-5) M; for the binding step K1 = 2.3(+/- 0.3) X 10(-3) M and the rate constants for the structural change are k2 = 26(+/-6)s-1 and k-2 = 0.61(+/- 0.04)s-1; the overall dissociation reaction enthalpy is delta H0 = 26(+/-6)KJmol-1 and the reactiom entropy is delta S0 = 4(+/- 20) kJ-1 mol-1. In combination with CD and X-ray crystallographic data, the results of this study suggest that the binding of the dipeptide to a trypsinogen-like, partially disordered conformation induces a transition to a trypsin-like highly ordered structure.

Animals↗
PubMed1996

Cloning and characterisation of a gene from Plasmodium vivax and P. knowlesi: homology with valine-tRNA synthetase.

We have previously described a lambdagt11 clone detected by immune screening with a monoclonal antibody (mAb) A12. This mAb is capable of completely blocking Plasmodium vivax transmission in the mosquito vector. An epitope recognised by A12 was mapped to six amino acids (aa) within the translated sequence of this clone. Here, we describe the complete sequence of the gene within which we mapped this epitope. Surprisingly, the translated sequence of the full-length open reading frame shows homology with that of valine-tRNA synthetases (Val-tRS) from other organisms. DNA cross-hybridisation with several of these species was observed by Southern blot. In addition, the corresponding gene has been obtained from the closely related simian malaria parasite, P. knowlesi. The two aa sequences show 66% identity and yet are very divergent from other Val-tRS sequences, apart from conserved blocks related to functional activity. Multiple sequence alignments reflect this dichotomy, as do predicted differences in antigenicity.

Amino Acid Sequence↗
PubMed1993

A mutant substituting valine for glycine at position 49 of Gs alpha induces neuronal differentiation of PC12 cells without activation of adenylate cyclase.

A GTPase-deficient mutant of the alpha-subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylate cyclase, substituting valine for glycine 49 (G49V) was transiently expressed in COS7 cells. The basal level of cAMP as well as an agonist-dependent accumulation of cAMP was two-fold higher in transfectants of Gs alpha (G49V) than in those of the normal counterpart. A stable transformant of PC12 cells expressing Gs alpha (G49V) under the control of a metallothionein promoter was then established. Two independent clones showed neurite outgrowth when the mutated Gs alpha was expressed by adding Cd2+ to culture medium. However, the level of basal cAMP of the transformant of PC12 cells with Gs alpha (G49V) was lower than that of the parental cells. The response to an agonist of the adenosine A2-receptor was suppressed in transformants. Although a cAMP-responsive element (CRE) was slightly activated by transfection and transient expression of Gs alpha in PC12 cells, no activation but a suppression of CRE was observed with PC12 cells transiently expressing Gs alpha (G49V). These results suggest that the mutant Gs alpha (G49V) couples adenylate cyclase in a different way in PC12 cells and may transduce differentiation signals through a cAMP-independent pathway.

Adenylyl Cyclases↗
PubMed2005

Lipophilic versus hydrogen-bonding effect in P3 on potency and selectivity of valine aspartyl ketones as caspase 3 inhibitors.

Caspase 3 is a cysteinyl protease that mediates apoptotic cell death. Its inhibition may have an important impact in the treatment of several degenerative diseases. The P1 aspartic acid residue is a required element of recognition for this enzyme that was maintained constant along with the adjacent natural valine as the P2 group. The thiobenzylmethylketone warhead on the aspartate was conveniently handled through solid-phase synthesis allowing modification in the P3 region that eventually led to simpler derivatives with increased potency against caspase 3. The key to such an effect is the introduction of hydroxyl group alpha to the P3 carbonyl.

Aspartic Acid↗
PubMed2005

Valine-286 residue in the third intracellular loop of the cholecystokinin 2 receptor exerts a pivotal role in cholecystokinin 2 receptor mediated intracellular signal transduction in human colon cancer cells.

Although expression of the gastrin/cholecystokinin-2 receptor (CCK2R) is widely reported in human colorectal cancer, little is known on its role in mediating mature amidated gastrin (gastrin-17 amide, G-17) induced intracellular signal transduction in colon cancer cells. The purpose of this study was to explore the intracellular events of colorectal cancer cells after gastrin binding to CCK2R. Meanwhile, the influence of a natural point mutation 286V-->F in the third intracellular loop of CCK2R on gastrin-envoked intracellular signal transduction was also investigated. Firstly, Colo320 cells were stably transfected with wild type (Colo320 WT) and mutant CCK2R (Colo320 M), respectively. The intracellular signal transduction events in response to gastrin were investigated in both Colo320 WT and Colo320 M cells. In Colo320 WT cells, G-17 induced formation of intracellular cyclic AMP and inositol 1,4,5-trisphosphate, and stimulated intracellular calcium mobilization. G-17 also stimulated tyrosine phosphorylation of ERKl/2, p38, FAK, and paxillin, and up-regulated the mRNA expression of early response gene c-Jun and c-Fos. However, G-17 inhibited proliferation and induced apoptosis in Colo320 WT cells. Mutation 286V-->F in the third intracellular loop of CCK2R blocked G-17 induced biological without affecting binding affinity of CCK2R to G-17. Our results suggest that activation of CCK2R by gastrin stimulates heterotrimeric G-protein Gq and G(12/13) mediated intracellular signal transduction pathway in colon cancer cells. The valine-287 residue in third intracellular loop of CCK2R plays a pivotal role in CCK2R mediated intracellular signal transduction.

Apoptosis↗