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beta-Chloro-L-alanine, an amino acid analog which inhibits a number of enzymes, reversibly inhibited the Escherichia coli K-12 alanine-valine transaminase, transaminase C. This inhibition, along with the inhibition of transaminase B, accounted for the isoleucine-plus-valine requirement of E. coli in the presence of beta-chloro-L-alanine.
Most of the coding sequence for the IlvN polypeptide subunit of acetohydroxyacid synthase I was deleted from the ilvB+ ilvN+ plasmid pTCN12 by in vitro methods. Several ilvB+ delta ilvN derivatives of pTCN12 were identified among transformants of a strain otherwise lacking any acetohydroxyacid synthase. Deletion derivatives produced an enzymatically active IlvB polypeptide, as shown by the Ilv+ phenotype of transformed cells and by immunologic and enzymatic assays. However, whereas the growth of pTCN12 transformants was sensitive to valine inhibition, growth of the ilvB+ delta ilvN transformants was relatively resistant. Moreover, in vitro analyses confirmed that both acetolactate and acetohydroxybutyrate synthesis in extracts of the ilvB+ delta ilvN transformants was resistant to valine inhibition, in comparison with that in extracts of pTCN12 transformants or with that catalyzed by purified acetohydroxyacid synthase I. The IlvN polypeptide had a minimal effect, if any, on IlvB polypeptide accumulation as measured by immunoprecipitation, but its absence resulted in a greater than 10-fold reduction in enzyme specific activity.
Tn5-induced mutants of Rhizobium meliloti that require the amino acids isoleucine and valine for growth on minimal medium were studied. In one mutant, 1028, the defect is associated with an inability to induce nodules on alfalfa. The Tn5 mutation in 1028 is located in a chromosomal 5.5-kb EcoRI fragment. Complementation analysis with cloned DNA indicated that 2.0 kb of DNA from the 5.5-kb EcoRI fragment restored the wild-type phenotype in the Ilv- Nod- mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to those for Escherichia coli IlvC and Saccharomyces cerevisiae Ilv5. Genes ilvC and ilv5 code for the enzyme acetohydroxy acid isomeroreductase (isomeroreductase), the second enzyme in the parallel pathways for the biosynthesis of isoleucine and valine. Enzymatic assays confirmed that strain 1028 was a mutant defective in isomeroreductase activity. In addition, it was shown that the ilvC genes of Rhizobium meliloti and E. coli are functionally equivalent. We demonstrated that in ilvC mutant 1028 the common nodulation genes nodABC are not activated by the inducer luteolin. E. coli ilvC complemented both defective properties (Ilv- and Nod-) found in mutant 1028. These findings demonstrate that R. meliloti requires an active isomeroreductase enzyme for successful nodulation of alfalfa.
A mutant of Corynebacterium glutamicum was found to accumulate high concentrations of a material which crystallized upon cooling of the broth. The compound was identified as tetramethylpyrazine. The mutant was found to require isoleucine, valine, leucine, and pantothenate for growth. All four requirements probably result from the loss of a single enzyme of the isoleucine-valine pathway. Since similar mutants of Neurospora crassa accumulate acetoin, the present mutant probably forms tetramethylpyrazine from acetoin. Accumulation of tetramethylpyrazine was dependent upon addition of thiamine. This observation is consistent with the known activity of diphosphothiamine as a cofactor for the formation of acetolactate (a precursor of acetoin) from pyruvate.
The role of valine aminoacylation of the two genomic RNAs of Peanut clump virus (PCV) was studied by comparing the amplification in vivo of RNAs with GAC, GDeltaC, or CCA anticodons in the tRNA-like structure (TLS) present at the 3' end of each viral RNA. The PCV RNA1 TLS of isolate PCV2 possesses a GAC anticodon and is capable of highly efficient valylation, whereas the RNA2 TLS has a GDeltaC anticodon that does not support valylation. The presence in RNA1 of GDeltaC or CCA anticodons that conferred nonvalylatability resulted in about 2- to 4-fold and a 14- to 24-fold reduction, respectively, in RNA accumulations in tobacco BY-2 protoplasts inoculated with the RNA1 variants together with wild-type RNA2(GDeltaC). No differences in RNA levels were observed among protoplasts inoculated with the three variant RNA2s in the presence of wild-type RNA1(GAC). All combinations of valylatable and nonvalylatable RNAs 1 and 2 were similarly infectious in Nicotiana benthamiana plants, and viral RNAs accumulated to similar levels; all input TLS sequences were present unchanged in apical leaves. In direct competition experiments in N. benthamiana plants, however, both RNA1 and RNA2 with GAC valylatable anticodons outcompeted the nonvalylatable variants. We conclude that valylation provides a small but significant replicational advantage to both PCV RNAs. Sequence analysis of the TLS from RNA2 of a second PCV isolate, PO2A, revealed the presence of an intact GAC valine anticodon, suggesting that the differential valylation of the genomic RNAs of isolate PCV2 is not a general characteristic of PCV.
The features of three babies with perinatal lethal osteogenesis imperfecta (OI II) resulting from substitutions of glycine by valine in the triple helical domain of the alpha 1(I) chain of type I collagen were studied. The babies were heterozygous for this substitution at residue 1006 in case 1 (OI35), 973 in case 2 (OI59), and 256 in case 3 (OI7B). OI35 had the most severe clinical form, OI IIC, with premature rupture of membranes, severe antepartum haemorrhage, stillbirth, severe short limbed dwarfism, and extreme osteoporosis. OI59 was a better formed baby but was also born prematurely as a result of premature rupture of membranes and severe antepartum haemorrhage. She had the radiographic features of OI IIA. OI7B was born at term and also had the radiographic features of OI IIA. Pathological examination of the skeletons of OI35 and OI59 showed grossly deficient intramembranous and endochondral ossification. Trabecular bone was sparse in the long bones and vertebrae. The trabeculae contained a cartilage core and an overlying layer of woven bone or osteoid. The diaphyses lacked cortical bone. The periosteal fibroblasts of OI35 contained grossly distended rough endoplasmic reticulum consistent with the 53% reduction in collagen secretion by cultured dermal fibroblasts. The aorta, skin, and lungs were hypoplastic in OI35 and OI59. The findings in this study show that glycine substitutions by valine in Gly-X-Y triplets, from glycine 256 to glycine 1006, of the triple helical domain of alpha 1(I) chains produce the OI II phenotype. The phenotype was most severe in the baby with the most carboxy-terminal substitution.
A man was studied with sporadic Creutzfeldt-Jakob disease (sCJD) who had serial cortical syndromes evolving over 15 months without significant ataxia, prominent myoclonus, or periodic complexes on EEG examinations. This clinical phenotype correlated with a predominantly cortical and striatal distribution of lesions and accumulation of protease resistant prion protein with relative sparing of the brainstem or cerebellum. No amyloid plaques were seen and prion protein (PrP) immunohistochemistry only demonstrated very faint granular deposits in the cerebral cortex. Molecular analysis showed homozygosity for valine at codon 129 in the prion protein gene (PRNP) and protease resistant prion protein type 1 deposition. The comparison of molecular and clinicopathological features of the present case with those previously reported in sCJD, indicates that valine homozygosity at codon 129 and type 1 protease resistant prion protein are associated with a distinct phenotypic variant of sCJD. The data also support the view that the PRNP codon 129 polymorphism and the physicochemical properties of the protease resistant prion protein are major determinants of phenotypic variability in sCJD.
Acetolactate synthase (ALS) specific activity was evaluated in isogenic lines of Saccharomyces cerevisiae carrying the wild-type ILV2 gene or mutations in this gene for resistance to the herbicide sulfometuron methyl (SM). Statistical comparisons were made between two nuclear alleles and among five alleles borne on a YE chimaeric plasmid transformed into a strain carrying a 1.5-kilobase deletion in the nuclear ILV2 gene. Decreased ALS activity of plasmid-borne SM-resistant mutations was shown not to be caused by copy number effects. ALS-specific activity in strains carrying the wild-type ILV2 allele exhibited strong feedback inhibition by valine and was sensitive to SM. All nuclear and plasmid-borne SM-resistance alleles resulted in ALS-specific activity highly resistant to SM and resistant to valine feedback inhibition.
Spiramycin biosynthesis in Streptomyces ambofaciens was stimulated in the presence of valine or by sequential addition of some short-chain fatty acids to a culture medium containing an ammonium salt as source of nitrogen. Acetate kinase and acetyl-CoA carboxylase, enzymes that catalysed the formation of precursors of spiramycin biosynthesis (acetyl-CoA and malonyl-CoA), were detected during the active growth and antibiotic production phases. In this latter phase a higher level of acetyl-CoA carboxylase activity was observed with valine (1.02 mumol.min-1.mg protein-1) than with ammonium (0.05 mumol.min-1.mg protein-1) as nitrogen source, while the evolution and the level of acetate kinase activity were the same in both media. Successive addition of acetate and isobutyrate stimulated highly and weakly the acetyl-CoA carboxylase and acetate kinase activity, respectively.
ATP is a potential marker of cell vialibility and growth. The content of ATP and 3H-valine incorporation into proteins were measured and the morphometry was performed after antidepressant treatment of astrocytes cultured in vitro with or without dibutyryl 3'5'-cyclic adenosine monophosphate (dB-cAMP). Antidepressants were added into the culture medium (for 24 h) at a final concentration of 10(-4)M (imipramine, amitriptyline, clomipramine, doxepine, mianserin) or 10(-5)M (maprotiline). It was shown that all antidepressants except maprotiline and imipramine increased ATP level and decreased 3H-valine incorporation into astrocytes. All drugs except clomipramine and maprotiline, diminished cell area and perimeter of astrocytes. The addition of dB-cAMP to cultures caused an increase of astrocyte form factor. It can be concluded that antidepressants have a significant effect on energy metabolism and differentiation of astrocytes cultured in vitro.
Primary cultures from fetal rat brain contain angiotensin II immunoreactivity in neurons. To study the origin of this immunoreactivity, primary cultures were incubated with [3H]-valine or [3H]-isoleucine. A time-dependent incorporation of radioactivity into immunoprecipitable angiotensin II was observed. Incubation of cultures with Captopril, an inhibitor of the converting enzyme, resulted in a significant inhibition of [3H]-valine incorporation into immunoreactive [3H]-angiotensin II. These results demonstrate that primary cultures from fetal rat brain can synthesize an angiotensin II-like immunoreactivity and support a concept that angiotensin II may be synthesized in fetal rat brain.
The intravenous application of the branched chain amino acid L-valine to patients with hepatic coma resulted in a specific drop of tryptophan, serotonin and 5-hydroxyindole acetic acid. Clinical improvement of hepatic encephalopathy and of the underlying neurotransmitter (serotonin) and ammonia derangements by administration of L-valine suggest a possible role of this competitive amino acid on intermediary metabolism and ammonia detoxification.
BACKGROUND: The sweet taste inhibitor lactisole acts on the human sweet taste receptor heteromer TAS1R2-TAS1R3 but not on its rodent counterpart. Recently, it was shown that the lactisole sensitivity of the human sweet taste receptor involves the part of TAS1R3 encompassing the seven transmembrane regions but not the huge N-terminal domain. Using mutational analysis we investigated which amino acid residues distinguish lactisole insensitive rat from sensitive human T1R3 receptors. RESULTS: The functional analysis of specific receptor mutants in HEK293T cells revealed that the exchange of valine 738 in the fifth transmembrane domain of rTas1r3 by an alanine is sufficient to confer lactisole sensitivity to the rat sweet taste receptor. The sensitivity of this receptor mutant is approximately 2 fold lower than the sensitivity of the human sweet taste receptor. Additional substitution of lysine 735 by phenylalanine in rTas1r3 results in a rat sweet taste receptor that is as sensitive to lactisole as its human counterpart. The exchange of valine 738 to alanine was accompanied by a approximately 50% reduction in receptor efficacy. This effect was seen with all six different sweet compounds examined. CONCLUSION: The lactisole insensitivity of rat sweet taste receptor is caused by only two amino acids in transmembrane region five, which is critical for the interaction of lactisole with the sweet taste receptor. The observation that the mutant receptor simultaneously displays a generally reduced sensitivity towards all agonists suggests that the lactisole insensitivity of the rodent receptor might be more likely caused by the inaccessibility of the lactisole binding site rather then by its direct disruption.
3-Hydroxyisobutyric aciduria, a disorder of valine metabolism, has been found in a boy in whom the clinical picture was that of a typical organic acidemia with repeated episodes of ketoacidosis requiring admission to hospital and parenteral fluid therapy, along with impressive failure to thrive and chronic lactic acidemia. The excretion of 3-hydroxyisobutyric acid ranged from 170 to 390 mmol/mol of creatinine. The administration of valine increased this to 18,700 mmol/mol of creatinine and reproduced the clinical picture of ketoacidosis. Concentrations of free carnitine were low, and esterified carnitine was elevated. Treatment with carnitine and a diet restricted in protein appeared to be beneficial.
The biosynthetic pathway of capsinoid in 'CH-19 Sweet' was investigated. [(3)H]Valine and [(14)C]phenylalanine were injected into the fruits of the intact plant. Both of radioactivities were detected in capsinoid fractions. (14)C radioactivity was observed in phenylpropanoid compounds, and in vanillin, vanillylamine, vanillyl alcohol, and vanillic acid. We confirmed that capsinoid is biosynthesized from phenylalanine and valine.
The influence of substrate concentrations on aminoacylation pathways and substrate specificities was investigated in the acylation reaction catalyzed by isoleucyl-tRNA synthetase from yeast. For the cognate substrates isoleucine and tRNAIle two Km values each differing by a factor about five were determined; the higher values were observed at concentrations higher than 1 microM, the lower values below 1 microM isoleucine or tRNAIle, respectively. At substrate concentrations below 1 microM also kcat values of the isoleucylation reaction are lowered. With the noncognate substrates valine and tRNAVal such differences could not be detected. The substrate ATP did not show any change of its Km value as far as the reaction was measurable. Under six different new assay conditions orders of substrate addition and product release followed sixtimes a sequential ordered ter-ter steady-state mechanism with ATP as the first substrate to be added, isoleucine as the second, and tRNAIle as the third one; pyrophosphate is the first product to be released, isoleucyl-tRNA the second, and AMP the third one. In one case this mechanism was modified by a rapid equilibrium segment for addition of ATP and isoleucine. From kcat and Km values and from AMP formation rates discrimination factors for discrimination between tRNAIleII and tRNAValI as well as between isoleucine and valine were determined. In the first case discrimination factors can vary up to a factor of thirty by changes of tRNA or amino-acid concentrations, in the second case discrimination factors are practically invariant. The two different Km values are hypothetically explained by assumption of anticooperativity in a flip-flop mechanism. Two hypothetical catalytic cycles are postulated.
Discrimination between isoleucine and valine is achieved with different accuracies by isoleucyl-tRNA synthetase from E. coli MRE 600. The recognition process consists of two initial discrimination steps and a pretransfer and a posttransfer proofreading event. The overall discrimination factors D were determined from kcat and Km values observed in aminoacylation of tRNA(Ile)-C-C-A with isoleucine and valine. From aminoacylation of the modified tRNA species tRNA(Ile)-C-C-A(3'NH2) initial discrimination factors I1 and pretransfer proofreading factors II1 were calculated. Factors I1 were computed from ATP consumption and D1, the overall discrimination in aminoacylation of the modified tRNA; factors II1 were calculated as quotient of AMP formation rates. Initial discrimination factors I2 and posttransfer proofreading factors II2 were determined from AMP formation rates observed in aminoacylation of tRNA(Ile)-C-C-A. The observed overall discrimination varies up to a factor of about four according to conditions. Under standard assay conditions 72,000, under optimal conditions 144,000 correct aminoacyl-tRNAs are produced per one error while 1.1 or 1.7 ATPs are consumed. A comparison with isoleucyl-tRNA synthetase from yeast shows that both enzymes act principally with the same recognition mechanism, but the enzyme from E. coli MRE 600 exhibits higher specificity and lower energy dissipation and does not show such high variation of accuracy as observed with the enzyme from yeast.
We describe a 67-year-old Japanese man with probable sporadic Creutzfeldt-Jakob disease (CJD) who had valine homozygosity at codon 129, a rarity in the Japanese. T2-weighted magnetic resonance imaging (MRI) detected high-intensity lesions in the bilateral middle cerebellar peduncles and basal ganglia as well as cerebellar and cortical atrophy. He developed cerebellar ataxia and subsequent mental deterioration, myoclonus, and periodic synchronous discharge as shown in an electroencephalogram. Cerebrospinal fluid examination showed a high level of neuron-specific enolase and a positive immunoassay for the 14-3-3 protein. He died of pneumonia 10 months after the initial symptoms appeared. Whether or not the genetic polymorphism increased his susceptibility to sporadic CJD is not clear because valine homozygosity at codon 129 is less than 1% in the normal Japanese population. Although there is no convincing evidence in the present case, the MRI findings of cerebellar peduncle changes, which are rare in CJD, suggest a kind of degeneration, demyelination, or both.