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PubMed1984

The valine, leucine and isoleucine requirements of white Leghorn chicks.

The requirements for leucine, isoleucine and valine were determined in 8- to 18-d-old White Leghorn male chicks. The requirements were estimated from body-weight gains. The estimated requirements for leucine, isoleucine and valine were 200, 115 and 125 mg/bird d, or 12.0, 5.0 and 8.0 g/kg diet, respectively. In terms of concentration in the diet, they are broadly similar to results for broiler-type chicks. When body-weight gain was plotted against daily intakes of each amino acid, significant (P less than 0.01) quadratic regression equations were obtained. The equations showed some characteristics of the individual amino acids.

Animals↗
PubMed1972

Separation of mitochondrial membranes of Neurospora crassa. II. Submitochondrial localization of the isoleucine-valine biosynthetic pathway.

Separation of Neurospora mitochondrial outer membranes from the inner membrane/matrix fraction was effected by digitonin treatment and discontinuous density gradient centrifugation. The solubilization of four isoleucine-valine biosynthetic enzymes was studied as a function of digitonin concentration and time of incubation in the detergent. The kinetics of the appearance of valine biosynthetic function in fractions outside of the inner membrane/matrix fraction, coupled with enzyme solubilization patterns similar to that for the matrix marker, mitochondrial malate dehydrogenase, indicate that the four isoleucine-valine pathway enzymes are localized in the mitochondrial matrix.

Alcohol Oxidoreductases↗
PubMed1979

Assessment of the essentiality of methionine, threonine, tryptophan, histidine and isoleucine in immature dogs.

A completely purified L-amino acid diet was used to evaluate the indispensability of methionine, threonine, tryptophan, histidine and isoleucine in the immature dog. Immature dogs fed diets devoid of methionine, threonine, tryptophan, histidine or isoleucine had severely depressed food intakes, accompanying weight loss, and a distinctly negative nitrogen balance. Partial or total removal of these amino acids resulted in a marked increase in blood and urinary urea. Estimates are made for the dietary requirements for these amino acids in the immature dog.

Alanine Transaminase↗
PubMed2002

Deficiency in methionine, tryptophan, isoleucine, or choline induces apoptosis in cultured cells.

Cells in culture die by apoptosis when deprived of the essential nutrient choline. We now report that cells (both proliferating PC12 cells and postmitotic neurons isolated from fetal rat brains) undergo apoptosis when deprived of other individual essential nutrients (methionine, tryptophan or isoleucine). In PC12 cells, deficiencies of each nutrient independently led to ceramide accumulation and to caspase activation, both recognized signals of several apoptotic pathways. A similar profile of caspases was activated in PC12 cells deprived of choline, methionine, tryptophan or isoleucine. More than one caspase was involved and these caspases appeared to transmit parallel signals for apoptosis induction because only broad-spectrum caspase inhibitors, but not inhibitors for specific individual caspases inhibited apoptosis in choline- or methionine-deprived cells. The induction of these caspase-dependent apoptosis pathways likely did not involve the same upstream signals. Choline deficiency perturbed choline metabolism but did not affect protein synthesis, whereas amino acid deficiencies inhibited protein synthesis but did not perturb choline metabolism. In addition, a subclone of PC12 cells that was resistant to choline deficiency-induced apoptosis was not resistant to tryptophan deficiency-induced apoptosis. These observations suggest that deficiency of each studied nutrient activates different pathways for signaling apoptosis that ultimately converge on a common execution pathway.

Amino Acids↗
PubMed1978

RNA synthesis in myeloma cells synchronized by isoleucine starvation.

Myeloma cells have been synchronized by isoleucine starvation. Changes in RNA synthetic rates as a result of starvation have been studied. The ability of isolated nuclei to synthesize RNA declines on starvation and increases subsequently on refeeding isoleucine. There is a coordinate drop in synthetic rate for all three polymerases both in vivo and in vitro. The chain elongation rate in vitro is the same in starved and normal cells, so the difference is in the number of active polymerases in vitro. However, the nuclei do not exactly parallel the state of the cell from which they were isolated, but the in vitro RNA synthesis increases more slowly than the in vivo RNA synthesis. There is no change in relative amounts of synthesis by the different RNA polymerases. The in vitro RNA product is similar in starved and growing cells.

Cell Division↗
PubMed1996

Characterization of a B. subtilis minor isoleucine tRNA deduced from tDNA having a methionine anticodon CAT.

Bacillus subtilis, which belongs to Gram-positive eubacteria, has been predicted to have a minor isoleucine tRNA transcribed from the gene possessing the CAT anticodon, which corresponds to methionine. We isolated this tRNA and determined its sequence including modified nucleotides. Modified nucleotide analyses using TLC, UV, and FAB mass spectroscopy revealed that the first letter of the anticodon is modified to lysidine [4-amino-2-(N6-lysino)-1-beta-d-ribofuranosyl pyrimidine]. As a result, this tRNA agrees with the minor one predicted from the DNA sequence and is thought to decode the isoleucine codon AUA.

Acylation↗
PubMed2002

Influence of supplemental lysine, isoleucine, threonine, tryptophan and total sulfur amino acids on egg weight of Hy-line W-36 hens.

Four experiments were conducted to determine whether synthetic lysine, isoleucine, threonine, and tryptophan (LITT) or TSAA supplementation to diets formulated based on lysine improved egg weight (EW) and hen performance. In Experiment 1, diets were formulated with three TSAA levels (0.65,0.72, and 0.81%). These diets were fed with and without adding lysine and isoleucine each at 0.055% and threonine and tryptophan each at 0.022%. Treatments were randomly assigned to 960 Hy-Line W-36 hens in eight replicates per treatment (20 birds/replicate). Inclusion of LITT improved (P < 0.05) EW within 2 wk with no TSAA x LITT interaction. Inclusion of LITT also increased average feed consumption (FC) by 1.4 g/d (P < 0.01). However, no effect (P > 0.05) of LITT was observed on egg production (EP), egg specific gravity (SG), or BW. The EW, EP, and FC increased linearly (P < 0.05) as dietary TSAA increased. The TSAA had no effect on SG or BW. In Experiment 2, supplemental LITT were removed to determine how rapidly hens lose the effect of LITT on performance. Within 2 wk, the effect of LITT on EW was lost. Average EW, EP, and FC of hens fed higher TSAA levels remained higher (P < 0.05) than hens fed 0.65% TSAA. In Experiment 3, all hens were fed a diet containing 0.65% TSAA to determine the time it takes to lose the effect of TSAA supplementation. The TSAA supplementation effect on EW, EP, and FC observed in the previous two experiments was lost within 1 wk. In Experiment 4, hens were again fed diets similar to Experiment 2 to confirm the time required for TSAA supplementation to improve EW. Within 1 wk, hens fed 0.72 or 0.81% TSAA improved (P < 0.05) EW over hens fed 0.65% TSAA. It was concluded that hens fed diets formulated based on lysine were deficient in LITT. Hens responded to the inclusion or removal of supplemental LITT and TSAA within 1 to 2 wk.

Amino Acids↗
PubMed2002

Isoleucine starvation caused by sulfometuron methyl in Salmonella typhimurium measured by translational frameshifting.

The authors have developed a tool for the study of inhibitor-induced amino acid starvation in bacteria which exploits the phenomenon of translational frameshifting. The inhibition of acetohydroxyacid synthase II by the herbicide sulfometuron methyl (SMM) has complex effects on branched-chain amino acid biosynthesis. Experiments were done with Salmonella typhimurium containing a plasmid with an isoleucine codon in a 'shifty' region, prone to translational frameshifting. SMM did not cause translational frameshifting in minimal medium under conditions that inhibit growth. A 20-fold higher concentration of SMM was required to cause starvation for isoleucine, e.g. in the presence of valine. This starvation was reflected in translational frameshifting correlated with inhibition of growth. These observations support the authors' previous suggestions based on other techniques. The method used here could be generalized for the study of complex metabolic effects related to amino acids.

Culture Media↗
PubMed2003

Metabolic engineering of valine- and isoleucine-derived glucosinolates in Arabidopsis expressing CYP79D2 from Cassava.

Glucosinolates are amino acid-derived natural products that, upon hydrolysis, typically release isothiocyanates with a wide range of biological activities. Glucosinolates play a role in plant defense as attractants and deterrents against herbivores and pathogens. A key step in glucosinolate biosynthesis is the conversion of amino acids to the corresponding aldoximes, which is catalyzed by cytochromes P450 belonging to the CYP79 family. Expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in Arabidopsis resulted in the production of valine (Val)- and isoleucine-derived glucosinolates not normally found in this ecotype. The transgenic lines showed no morphological phenotype, and the level of endogenous glucosinolates was not affected. The novel glucosinolates were shown to constitute up to 35% of the total glucosinolate content in mature rosette leaves and up to 48% in old leaves. Furthermore, at increased concentrations of these glucosinolates, the proportion of Val-derived glucosinolates decreased. As the isothiocyanates produced from the Val- and isoleucine-derived glucosinolates are volatile, metabolically engineered plants producing these glucosinolates have acquired novel properties with great potential for improvement of resistance to herbivorous insects and for biofumigation.

Arabidopsis↗
PubMed1975

The effects of spermine and Mg2+ on the catalytic mechanism of isoleucine: tRNA ligase.

Isoleucyl-tRNA formation catalysed by isoleucine: tRNA ligase is stimulated by both Mg2+ and spermine in the pH-range 7.0 to 8.0 at 310 K. At low [Mg2+] the acceleration caused by both cations together exceeds the sum of their individual effects. 2. The spermine-stimulated reaction has a steeper temperature-dependence than reaction in the presence of Mg2+. Two phases in the kinetics of isoleucyl-tRNA formation are detected in the presence of Mg2+ plus or minus spermine, but only a single step is observed in the presence of spermine alone. Thus the rate-limiting steps under normal assay conditions are different for the two cations. 3. Enzyme-bound isoleucyl-AMP can be formed in the absence of Mg-2+ and plus or minus spermine. 4. It is concluded that there is no evidence for cation-dependent differences in the reaction mechanism of isoleucine: tRNA ligase, though there are certainly differences in the relative rates of some of the individual steps.

Enzyme Activation↗
PubMed1984

Effects of mitochondrial protein synthesis inhibitors on the incorporation of isoleucine into Plasmodium falciparum in vitro.

Plasmodium falciparum was grown in human erythrocytes in vitro and the effect of chloramphenicol, erythromycin, and tetracycline on growth and maturation of the parasites and on their ability to incorporate [3H]isoleucine into protein was observed. Exposure of rings to high concentrations of chloramphenicol had little effect on subsequent maturation of the rings whereas brief (4 h) exposure of trophozoites caused a dose-dependent inhibition of subsequent ring formation. Incorporation of [3H]isoleucine into protein was not affected during at least 6 h of exposure to high concentration of the three drugs examined, but appreciable inhibition was observed after 21 h, with chloramphenicol being the least effective inhibitor. These results suggest that there is a stage-specific effect of inhibition of mitochondrial protein synthesis on subsequent development and that the mitochondria are essential for growth and development even though they lack a functional Krebs cycle.

Animals↗
PubMed1995

Decreased plasma alanine and isoleucine in Huntington's disease.

Amino acid concentrations in plasma of patients with Huntington's disease (HD) were determined in 16 patients and 21 age- and sex-matched healthy controls. Alanine and isoleucine were significantly decreased in HD plasma whereas arginine, histidine, leucine, lysine, ornithine, proline, serine, threonine, tyrosine, and valine showed no significant changes. Our findings confirm the decreases of alanine and isoleucine that were described in plasma and cerebrospinal fluid by other investigators. A possible defect in cellular uptake or metabolism of neutral amino acids seems to be a consistent feature of HD.

Adult↗
PubMed1996

Corneal deepithelialization caused by acute deficiency of isoleucine during treatment of a patient with maple syrup urine disease.

Classical Maple Syrup Urine Disease is a serious, autosomal recessive and rare metabolic disorder due to a completely inactive enzyme complex for metabolizing the branched amino acids leucine, valine and isoleucine. If untreated the disease is lethal. Metabolic control achieved by strict dietary treatment results in normal development. In this paper we describe deepithelialization of the cornea together with skin and intestinal symptoms as the result of isolated deficiency of isoleucine during treatment of a critically ill, newborn infant with this unusual disease.

Acute Disease↗
PubMed1977

The influx of isoleucine into the cerebral hemispheres and cerebellum: carrier-mediated transport and diffusion.

The influx of L-isoleucine from the blood into the cerebral hemispheres, and into the cerebellum, of the living rat was studied. When various raised levels of L-isoleucine were maintained in the circulation, influx did not increase in proportion to the raised blood levels, which showed that a carrier-mediated transport was being saturated. It was found that the values for influx could not be explained precisely by Michaelis-Menten kinetics. The divergence of the observed values for influx, from those which were obtained by the application of Michaelis-Menten kinetics, could be explained if part of the influx was due to a small, non-saturable component. This minor component of influx may be due to diffusion or to a second carrier-mediated transport system that is not readily saturated.

Animals↗
PubMed1986

L-isoleucine and L-leucine: tumor promoters of bladder cancer in rats.

A 4-week assay for screening tumor promoters of bladder cancer has been developed in which increased agglutinability of isolated rat bladder cells with concanavalin A is used as an indicator. On the basis of this assay system, L-isoleucine and L-leucine were suspected of being possible tumor promoters. Results of 40- to 60-week carcinogenesis experiments in which N-butyl-N-(4-hydroxybutyl)nitrosamine was used as an initiator demonstrate that L-isoleucine and L-leucine promote bladder cancer in rats. This finding may be relevant to the high incidence of human bladder cancer in Western countries, where the diet is rich in protein.

Animals↗
PubMed1971

Salmonella typhimurium mutants with alternate requirements for vitamin B 6 or isoleucine.

Several mutants of Salmonella typhimurium LT-2, isolated as auxotrophs for vitamin B(6), grew without the added vitamin when supplied with either isoleucine, alpha-ketobutyrate, or alpha-keto-beta-methylvalerate, but not with threonine or with other alpha-keto acids. When grown on minimal medium supplemented with isoleucine, these mutants synthesized vitamin B(6) in amounts comparable to wild-type cells; they thus appeared to contain a modified l-threonine dehydratase and to belong to genotype ilvA (threonine dehydratase) instead of pdx (pyridoxine). Direct assays confirmed this hypothesis. Wild-type cells (toluene-treated) showed approximately the same threonine dehydratase activity whether grown in the presence or absence of added pyridoxal-P; mutant cells approached the activity of wild-type cells only when they were grown with added vitamin B(6) and were assayed in the presence of pyridoxal-P. In cell-free extracts, the threonine dehydratase from mutant cells was cold labile and more labile to oxidative inactivation than the wild-type enzyme; furthermore, activation of the mutant apoenzyme required a 10- to 20-fold higher concentration of pyridoxal-P than was required for the wild-type apoenzyme. These results show that cultures which appear auxotrophic for a given vitamin may synthesize that vitamin in normal amounts, the exogenous requirement arising from impaired binding of the vitamin-derived coenzyme to a genetically altered apoenzyme dependent on that coenzyme. Inadequate nutritional data to support the genetic findings can lead to erroneous genotype classification for such mutants.

Amino Acids↗
PubMed1974

Derepressed levels of the isoleucine-valine and leucine enzymes in his T 1504, a strain of Salmonella typhimurium with altered leucine transfer ribonucleic acid.

A hisT mutant of Salmonella typhimurium was found to have altered regulation of the isoleucine-valine and leucine enzymes. These enzymes in the hisT strain were derepressed two- to eightfold over those of the parent wild-type strain when grown in minimal medium or under repressing conditions. The amount of tRNA(Leu) and the cellular concentration of charged tRNA(Leu) was about the same in the hisT strain and in the wild type. However, leucyl-tRNA from the mutant was chromatographically different from that of wild type, confirming previous reports that hisT strains have altered tRNA(Leu). These results suggest strongly that tRNA(Leu) is involved in repression of the isoleucine-valine and leucine enzymes in S. typhimurium.

Carbon Radioisotopes↗