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Electrospray ionization mass spectra of lead(II) nitrate-amino acid mixtures enable unequivocal identification of lead complexes for each of the essential amino acids and a valine complex is reported as the first crystallographically characterized lead-amino acid complex.
1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.
G.l.c.--mass-spectral analysis of headspace above cultures of Aeromonas and Moraxella spp. indicates the presence of isobutyronitrile, isobutyraldoxime O-methyl ether, methacrylonitrile and possibly methacrylaldoxime O-methyl ether. Accumulation of these catabolites is maximal under low oxygen concentrations and is enhanced by enrichment of the medium with valine. Isobutyraldoxime O-methyl ether is established as the compound observed but not identified in previous studies with other bacterial species involved in spoilage of meat and chicken.
A chemical cleavage method for detecting mismatched bases in heteroduplexes formed between patient mRNA and control cDNA probes was employed to identify a single base mutation in a heterozygous case of osteogenesis imperfecta type IV. The parents' fibroblast mRNA did not contain the mutation. The region of the mRNA mismatch was amplified by using the polymerase chain reaction, cloned and sequenced. A point mutation of G to U at base-pair 2162 of the collagen alpha 2(I) mRNA resulted in the substitution of glycine by valine at amino acid position 586 of the helix. This substitution disrupted the critical Gly-Xaa-Yaa repeating unit of the collagen triple helix and resulted in helix destabilization, as evidenced by a decreased thermal stability. This local disturbance to helix propagation from the C-terminus to the N-terminus led to the overmodification of the collagen helix downstream towards the N-terminus. However, collagen secretion in vitro was normal, and the clinical phenotype probably resulted from the secretion into the extracellular matrix of the mutant collagen combined with a decrease in collagen production to 65% of control values. The rapid detection of the osteogenesis imperfecta mutation by using the chemical cleavage method afforded the opportunity to apply the technique to prenatal diagnosis in the next pregnancy of the mother of the osteogenesis imperfecta patient. The absence of a mismatched base in chorionic villus mRNA and control cDNA heteroduplexes indicated that the foetus did not carry the mutation, which was confirmed by the subsequent delivery of a normal baby.
In order to determine the requirement of lactating sows for valine (Val) the effects of graded Val supplementations on feed intake, milk yield and changes of body weight of sows and piglets were examined using 54 multiparous sows over a total of 72 lactations (35 days). The sows were randomly assigned to six treatments (1-6). Each group received the same basic diet, covering the requirements of energy and nutrients except Val (4.5 g native Val, i.e. 3.5 g apparent ileal digestible Val). Adding crystalline l-Val dietary Val concentrations of 5.5, 6.5, 8.5, 10.5 and 14.5 g/kg, respectively, were realized. Feed intake of the sows was determined daily, body weight was determined on day 110 of gestation and day 1, 22 and 35 of lactation. The piglets were weighed on day 1, 21 and 35. The intake of piglet feed, which was offered from day 21, was determined at day 35 for each litter. Milk yield was determined using the weigh-suckle-weigh-method at day 13, 14, 20 and 21, fat and protein contents were analysed in hand-milked samples with infrared spectroscopy. Feed intake (kg/day) of the sows during lactation averaged 2.9, 4.3, 4.6, 4.6, 4.9 and 5.1 for treatments 1-6, respectively. For treatments 1 and 2 body weight losses of the sows during lactation were higher (1207 and 805 g/day, respectively) and milk production was lower (7076 and 8686 g/day, respectively), compared with the average of treatments 3-6 (625 g/day body weight loss; 9263 g milk/day). Daily weight gain of the piglets was also lower (146 and 171 g/day vs. 200 g/day) leading to lower weaning weights of 6.6 and 7.5 vs. 8.5 kg. As a consistent growth depression of the piglets in Val deficiency must be assumed, at least 6.5 g Val corresponding to 5.5 g apparent ileal digestible Val per kg lactation feed must be recommended.
Recently, 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase; MTH1), a key enzyme for preventing oxidative stress-induced DNA damage, has been found to be expressed aberrantly in the nigrostriatal dopaminergic neurones in the brains of those with Parkinson's disease (PD). A valine (Val) to methionine (Met) polymorphism at codon 83 in exon 4 of the MTH1 gene was studied in 73 patients with sporadic PD and 151 age-matched non-PD controls by PCR-RFLP analysis, to determine a possible association of this polymorphism with development of PD. The frequency of either 83Val or 83Met allele was not statistically different between PD patients (92.5% or 7.5%) and the controls (88.7% or 11.3%) (chi(2) = 1.511, P = 0.2190). The 83Met/Met homozygotes consisting of an infrequent genotype in the control population (1.3%) were not found in the PD group. The frequency of both 83Val/Met heterozygotes and 83Met/Met homozygotes combined was not statistically different between PD patients (15.1%) and the controls (21.2%), compared with that of the 83Val/Val homozygotes (chi(2) = 1.190, P = 0.2754). These results indicate that the 83Val/Met polymorphism in the MTH1 gene is not associated with an increased risk for development of sporadic PD.
Data are presented suggesting that intact Neurospora crassa mitochondria contain an enzyme complex incorporating five enzymes necessary for the biosynthesis of isoleucine and valine. The functional integrity and stability of the complex has been shown to be dependent on the metabolic state of the mitochondria. The complex has been solubilized by digitonin and has an approximate molecular weight of 400,000.
Minihelices mimicking the amino acid acceptor and anticodon branches of yeast tRNA(Val) have been synthesized by in vitro transcription of synthetic templates. It is shown that a minihelix corresponding to the amino acid acceptor branch and containing solely a valine-specific identity nucleotide can be aminoacylated by yeast valyl-tRNA synthetase. Its charging ability is lost after mutating this nucleotide. This ability is stimulated somewhat by the addition of a second hairpin helix that mimicks the anticodon arm, which suggests that information originating from the anticodon stem-loop can be transmitted to the active site of the enzyme by the core of the protein.
We present the first steps in applying transverse relaxation-optimized spectroscopy (TROSY) techniques to the study of allosterism. Each beta-chain of the hemoglobin (Hb) tetramer has 17 valine residues. We have (15)N-labeled the beta-chain Val residues and detected 16 of the 17 (1)H-(15)N correlation peaks for beta-chain Val of the R state CO-Hb structure by using the TROSY technique. Sequence-specific assignments are suggested, based mainly on analysis of the (1)H pseudocontact-shift increments produced by oxidizing the diamagnetic R state HbCO to the paramagnetic R state fluoromet form. When possible, we support these assignments with sequential nuclear Overhauser effect (NOE) information obtained from a two-dimensional [(1)H,(1)H]-NOESY-TROSY experiment (NOESY, NOE spectroscopy). We have induced further the R-T conformational change by adding the allosteric effector, inositol hexaphosphate, to the fluoromet-Hb sample. This change induces substantial increments in the (1)H and (15)N chemical shifts, and we discuss the implication of these findings in the context of the tentative sequence assignments. These preliminary results suggest that amide nitrogen and amide proton chemical shifts in a selectively labeled sample are site-specific probes for monitoring the allosteric response of the ensemble-averaged solution structure of Hb. More important, the chemical-shift dispersion obtained is adequate to permit a complete assignment of the backbone (15)N/(13)C resonances upon nonselective labeling.
Escherichia coli acyl carrier protein (ACP) has been reported to exist in at least two distinct conformers in solution. A novel form of ACP having an increased electrophoretic mobility on polyacrylamide gel electrophoresis was noted previously during work on beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants of E. coli (Jackowski, S., and Rock, C. O.(1987) J. Bacteriol. 169, 1469-1473). These workers reported that the increased electrophoretic mobility of the ACP from fabF strains occurred irrespective of prosthetic group attachment or the state of acylation of the prosthetic group. Since these workers were unable to detect a difference between the amino acid sequence of the ACP from the fabF mutants and that of wild type ACP, they suggested that the increased electrophoretic mobility was due to an unknown post-translational modification of the polypeptide chain. We have reinvestigated these mutants and report that the increased electrophoretic mobility is due to a mutation within the gene (acpP) that encodes ACP. This mutation results in substitution of isoleucine for valine 43 of ACP. Site-directed mutagenesis of a synthetic ACP gene demonstrated that the amino acid substitution at residue 43 is the cause of the increased electrophoretic mobility. Gel filtration experiments indicated that the increased electrophoretic mobility results from the more compact structure of V43I ACP at high pH. The altered residue lies within the ACP region of greatest conformational lability, and thus the V43I substitution may shift the equilibrium toward the more compact conformation(s). The disulfide-linked dimer of V43I ACP was readily formed and had an electrophoretic migration greater than the dimer of wild type ACP, suggesting that formation of ACP.ACP dimers does not require structural deformation of the protein.
The amino acid change V75T in human immunodeficiency virus type 1 reverse transcriptase confers a low level of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine, d4T) resistance in vivo and in vitro. Valine 75 is located at the basis of the fingers subdomain of reverse transcriptase between the template contact point and the nucleotide-binding pocket. V75T reverse transcriptase discriminates 3.6-fold d4T 5'-triphosphate relative to dTTP, as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. In addition, V75T increases the DNA polymerization rate up to 5-fold by facilitating translocation along nucleic acid single-stranded templates. V75T also increases the reverse transcriptase-mediated repair of the d4TMP-terminated DNA by pyrophosphate but not by ATP. The V75T/Y146F double substitution partially suppressed both increases in rate of polymerization and pyrophosphorolysis, indicating that the hydroxyl group of Thr-75 interacts with that of Tyr-146. V75T recombinant virus was 3-4-fold d4T-resistant and 3-fold resistant to phosphonoformic acid relative to wild type, confirming that the pyrophosphate traffic is affected in V75T reverse transcriptase. Thus, in addition to nucleotide selectivity V75T defines a type of amino acid change conferring resistance to nucleoside analogues that links translocation rate to the traffic of pyrophosphate at the reverse transcriptase active site.
We have combined Fourier transform infrared/temperature derivative (FTIR-TDS) spectroscopy at cryogenic temperatures and flash photolysis at ambient temperature to examine the effects of polar and bulky amino acid replacements of the highly conserved distal valine 68 in sperm whale myoglobin. In FTIR-TDS experiments, the CO ligand can serve as an internal voltmeter that monitors the local electrostatic field not only at the active site but also at intermediate ligand docking sites. Mutations of residue 68 alter size, shape, and electric field of the distal pocket, especially in the vicinity of the primary docking site (state B). As a consequence, the infrared bands associated with the ligand at site B are shifted. The effect is most pronounced in mutants with large aromatic side chains. Polar side chains (threonine or serine) have only little effect on the peak frequencies. Ligands that migrate toward more remote sites C and D give rise to IR bands with altered frequencies. TDS experiments separate the photoproducts according to their recombination temperatures. The rates and extent of ligand migration among internal cavities at cryogenic temperatures can be used to interpret geminate and bimolecular O2 and CO recombination at room temperature. The kinetics of geminate recombination can be explained by steric arguments alone, whereas both the polarity and size of the position 68 side chain play major roles in regulating bimolecular ligand binding from the solvent.
Three isoforms of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) are known to exist in mammalian cells. This study investigated the effects of thapsigargin and a variety of commonly used hydrophobic inhibitors on these SERCA isoforms (i.e. SERCA1b, SERCA2b, and SERCA3a), which were transiently expressed in COS-7 cells. In addition, the study assessed whether the introduction of the phenylalanine to valine mutation at position 256 (F256V), known to reduce the potency of thapsigargin inhibition in avian SERCA1, affects the other SERCA isoforms in a similar manner and whether this mutation also affects the inhibition by other inhibitors. This study has shown that the sensitivity to thapsigargin is different for the SERCA isoforms (apparent K(i) values being 0.21, 1.3, and 12 nm for SERCA1b, SERCA2b, and SERCA3a, respectively). The reduction in thapsigargin sensitivity caused by the F256V mutation was also different for the three isoforms, with SERCA2b only being modestly affected by this mutation. Although some of the other inhibitors investigated (i.e. cyclopiazonic acid and curcumin) showed some differences in their sensitivity toward the SERCA isoforms, most were little affected by the F256V mutation, indicating that they inhibit the Ca(2+)-ATPase by binding to sites on SERCA distinct from that of thapsigargin.
Four experiments were conducted to evaluate valine (Val) requirements in Ross 508 broilers from 3 to 6 weeks of age. Common diets were fed to broilers until 3 weeks of age. Growth and carcass measurements were taken in all experiments. Immune responsiveness measurements were taken in Experiments 1 and 2. Birds given 7.2 g Val/kg of diet in Experiment 1 had more abdominal fat than birds given 8.2 g Val/kg of diet, but there were no differences in growth or other carcass measurements. Because growth performance was not reduced in birds given 7.2 g Val/kg of diet, Val concentration was reduced to 6.4 g Val/kg of diet in Experiments 2 and 3. Increasing Val from 6.4 to 8.7 g Val/kg of diet resulted in linear increases for BW gain, feed efficiency and Val intake in male birds, and Val intake in female birds. Quadratic responses to increasing dietary Val were not observed in any experiment. There were no effects of Val on innate or adaptive immunity. A nonessential amino acid mixture containing the same nitrogen content as the L-Val additions in Experiment 4 was added and tends to support the idea that the responses to Val were specific and not due to increases in total nitrogen. Dose responses to Val resulted in male, but not female, birds given 7.3 g Val/kg of diet having improved BW gain and feed efficiency compared with birds receiving 6.4 g Val/kg of diet.
Propylene oxide (PO) is an important industrial compound and a directly acting mutagen. Human exposure to PO can be monitored by the determination of haemoglobin adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxypropyl)valine in whole haemoglobin was developed and its potential usefulness as a tool for biologically monitoring occupational exposure was demonstrated. Analytical reliability was confirmed in a comparative study with GC-MS (range 3.7-992 nmol g-1 haemoglobin (Hb), correlation coefficient 0.99, n=10). The assay has been configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay employs a whole blood matrix and has a working range of 2-250 pmol g-1 Hb. It does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results in potentially exposed workers indicate the assay's high potential usefulness in routine occupational biomonitoring of exposure to PO.
Ten affected individuals are described from a kindred with autosomal dominant familial Alzheimer's disease in which a mutation in the amyloid precursor protein gene results in a valine to glycine substitution at amyloid precursor protein 717 which co-segregates with the disease. The mean age at onset of symptoms was 52 years with a range from 40 years to 67 years. The median duration of the disease was 11 years, with a range of 7-16 years. All individuals fulfilled the National Institute for Neurological and Communicative Disorders and Stroke criteria for probable Alzheimer's disease. A homogeneous clinical and neuropsychological pattern was evident within the family. Myoclonic jerks, seizures, depression and a lack of insight were common features. Positron emission tomography demonstrated biparietal bitemporal hypometabolism in the one affected individual who was studied. The diagnosis was confirmed histopathologically in one individual.
Stickler syndrome (hereditary arthro-ophthalmopathy) is the commonest inherited cause of retinal detachment and one of the commonest autosomal dominant connective tissue dysplasias. There is clinical and locus heterogeneity with about two thirds of families linked to the gene encoding type II procollagen (COL2A1). Families with Sticklers syndrome type 1 have a characteristic congenital vitreous anomaly and are linked without recombination to markers at the COL2A1 locus. In contrast families with the type 2 variety have a different vitreo-retinal phenotype and are not linked to the COL2A1 gene. Type XI collagen is a quantitatively minor fibrillar collagen related to type V collagen and associated with the more abundant type II collagen fibrils. A mutation in COL11A2, the gene for alpha 2 (XI) procollagen, has recently been found in a family described as having Stickler syndrome, although there was no ocular involvement. Here we show for the first time that a family with the full Type 2 Stickler syndrome including vitreous and retinal abnormalities is linked to the COL11A1 gene and characterise the mutation as a Glycine to Valine substitution at position 97 of the triple helical domain caused by a single base G-->T mutation. These results are the first to provide confirmation that type XI collagen is an important structural component of human vitreous. They also support previous work suggesting that mutations in the genes encoding collagen XI can give rise to some manifestations of Stickler syndrome, but of these, only mutations in COL11A1 will give the full syndrome including the vitreo-retinal features.
Several experiments were conducted to quantitatively evaluate the growth-promoting capacity of the isomers of the branched-chain amino acids (BCAA) and their alpha-hydroxy and alpha-keto analogs for the chick and rat. Basal chemically defined diets were formulated to be singly deficient in the BCAA under study; analogs therefore were evaluated as sources of supplemental amino acid activity. DL-Isoleucine (DL-Ile), alpha-keto-beta-L-methylvaleric acid (KMV) and the isomers of alpha-hydroxy-beta-methylvaleric acid (HMV) were inferior to an isomolar level of L-Ile and varied considerably in their capacity to support growth of chicks and rats. Efficacy values of the compounds for chicks (C) and rats (R) were: DL-Ile 85% (C), 58% (R); KMV 60% (C), 38% (R); L-HMV 84% (C), 65% (R); DL-HMV 49% (C), 45% (R); and D-HMV -10% (C), -2% (R). The isomers of valine (Val) and alpha-hydroxyisovaleric acid (HIV) had efficacy values in chicks of: DL-Val, 84%; D-Val, 72%; L-HIV, 82%; DL-HIV, 79%; D-HIV, 66%. D-Val had only marginal growth-promoting activity for rats, 16%. All Val analogs had comparable efficacy values for the rat: alpha-ketoisovaleric acid (KIV), 49%; L-HIV, 54%; DL-HIV, 51%; and D-HIV, 46%. DL-Leu and D-Leu, alpha-ketoisocaproic acid (KIC), and L-, DL- and D-alpha-hydroxyisocaproic acid (HIC) were all less efficacious than isomolar amounts of supplemental L-Leu in rats. Efficacy values were: DL-Leu, 78%; D-Leu, 48%; KIC, 56%; L-HIC, 58%; DL-HIC, 55%; and D-HIC, 41%. The BCAA keto analogs were not superior to the L-alpha-hydroxy analogs in growth-promoting capacity, and this is of great interest for the potential use of these nitrogen-free amino acid analogs in nutritional therapy.