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At least 199 records · Page 11Linked to original sources
PubMed1978

Differing effects of isoleucine deficiency on the toxicity of MNNG for 10T1/2 and CHO cells.

Isoleucine deficiency sensitizes C3H 10T1/2 cells to the cytotoxic effects of MNNG. Synchrony of proliferation is not required for this effect since it occurs prior to full growth arrest and subsequent establishment of synchronous proliferation after refeeding in complete medium. Furthermore, confluence arrest of proliferation of 10T1/2 cells does not affect their cytotoxic response to MNNG, although they proliferate synchronously after replating at low density. In contrast, the toxicity of MNNG for CHO cells is not altered by isoleucine deficiency, even though these cells are readily synchronized by refeeding in complete medium after transient isoleucine deficiency.

Cell Count↗
PubMed1994

Expression of Na+-independent isoleucine transport activity from rat brain in Xenopus laevis oocytes.

Poly(A)+ RNA from C6-BU-1 rat glioma cells and rat astroglial cells induced isoleucine transport activity when injected into Xenopus laevis oocytes. The Na+-independent component of isoleucine transport was inhibited by leucine, phenylalanine and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) but neither by methylaminoisobutyric acid (MeAIB) nor lysine. A Km value of approx. 100 microM was determined for the Na+-independent transport of isoleucine. These data are in accordance with expression of a system L like transporter. By injection of size fractionated poly(A)+ RNA a length of approx. 1.9 kb was determined for the pertinent mRNA.

Amino Acid Transport Systems↗
PubMed1985

Control of isoleucine-valine biosynthesis in a valine-resistant mutant of Escherichia coli K-12 that simultaneously acquired azaleucine-resistance.

A mutant of Escherichia coli K-12 isolated as being growth resistant to L-valine (Valr) was shown also to exhibit growth resistance to 4-azaleucine (Azlr). Transductional analysis indicated that Azlr is cotransduced with Valr at a frequency of 100% and both are linked to leu, ara, and carA. This mutation conferring valine and azaleucine growth resistance resulted in increased levels of isoleucine and valine biosynthetic enzymes as well as those of valyl- and isoleucyl-tRNA synthetases during growth in minimal and enriched media. Acquisition of Vals/Azls results in the restoration of normal regulation of both classes of ilv enzymes and normal patterns of the tRNA Ile species. The overall regulatory patterns observed for individual isoleucine and valine gene products suggest differential participation of isoleucine and valine and/or isoleucyl- and valyl-tRNA's in control of expression of the respective structural genes.

Acetolactate Synthase↗
PubMed1985

Location of the isoleucine arrest point in CHO and 3T3 cells.

An isoleucine arrest point in G1 was determined by two methods for CHO and 3T3 cells. In the first method the fraction of cells entering S after isoleucine deprivation was assessed by [3H]thymidine labelling and autoradiography. In the second method cells entering S after isoleucine deprivation were identified by double-label autoradiography using [3H] and [14C]thymidine. From the fraction of cells entering S, determined by the two methods, the arrest point in G1 (and entry into G0) is located within the last 40 min of G1.

Animals↗
PubMed1985

Incorporation of isoleucine into protein by a soluble fraction from spermatozoa.

A heat-labile, non-dialyzable factor(s) in soluble fractions from porcine, bull, rabbit and cock spermatozoa was found to incorporate the radioactivity of [14C]isoleucine into a 95 degrees C CCl3COOH-insoluble fraction. The incorporation required ATP, Mg2+, casein and 2-mercaptoethanol. Trypsin and alpha-chymotrypsin inhibited the incorporation, while RNase A and DNase I did not. A mixture of 19 amino acids other than isoleucine had no effect on the incorporation. The reaction product was identified as protein. The incorporated moiety was the isoleucyl moiety of isoleucine and it retained a free alpha-amino group in the product protein. Some other characteristics of this incorporation are also described.

Animals↗
PubMed1995

Induction of volatile biosynthesis in the lima bean (Phaseolus lunatus) by leucine- and isoleucine conjugates of 1-oxo- and 1-hydroxyindan-4-carboxylic acid: evidence for amino acid conjugates of jasmonic acid as intermediates in the octadecanoid signalling pathway.

One of the most intriguing plant defense reactions against herbivores is the emission of volatiles as potentially attractive signals for the natural enemies of the attacking species. Like many other low and high molecular weight chemical defenses, volatile production is under the control of the octadecanoid signalling pathway leading to jasmonic acid (2) (threshold concentration of jasmonic acid giving rise to volatile induction in Phaseolus lunatus: approximately 100 nmol.ml-1). A significantly more active compound is the phytotoxin coronatine (3) (threshold concentration: > or = 1 nmol.ml-1). Methyl esters of 1-oxo-indanoyl-isoleucine (4) or 1-oxo-indanoyl-leucine (5), designed as readily available analogues of coronatin (3), have also been shown to be active (threshold concentration: > or = 20 nmol.ml-1). Crucially, their component parts, i.e. 1-oxo-indan-carboxylic acid and the amino acids are completely inactive. The pattern of emitted volatiles, produced by plants treated with these analogues, is largely identical to that released from coronatine- or jasmonic acid-treated plants. While the reduction of the carbonyl group of jasmonic acid (2) results in an inactive molecule, namely curcurbic acid, the methyl ester of the 1-hydroxy-indanoyl-isoleucine conjugate (8) is at least as effective as the corresponding oxo-derivatives (4) and (5) (threshold concentration: > or = 20 nmol.ml-1). The results support the concept that epi-jasmonic acid (1) may be converted into a leucine or isoleucine conjugate at an early stage in the natural signal transduction pathway. Their later interaction with a macromolecular receptor apparently requires enolization of the carbonyl group in the jasmonate moiety, yielding a planar segment which is essential for successful binding with the macromolecule. The resulting hydroxy group is implicated in the formation of a hydrogen bond in the ensuing ligand/receptor complex.

Amino Acids↗
PubMed1983

Release of leucine and isoleucine metabolites by perfused skeletal muscle and liver of rat.

The oxidation of leucine, isoleucine and the corresponding 2-ketoacids was studied in perfused hindquarter and liver of rat. The metabolites from these compounds released into the perfusate were identified by anion exchange chromatography and gas chromatography/mass spectrometry. Muscle perfused in the presence of leucine accumulated ketoleucine (2-ketoisocaproate) and isovalerate, while perfusion with isoleucine resulted in ketoisoleucine (2-keto-3-methylvalerate) and 2-methylbutyrate. Octanoate stimulated the oxidation of the branched-chain amino acids. The fatty acid did not change the amount of branched-chain 2-keto acids released into the perfusate, but increased the output from the muscle of the oxidation products isovalerate (from leucine) and 2-methylbutyrate (from isoleucine). Rat liver perfused with ketoleucine released mainly ketone bodies and small quantities of 3-hydroxyisovalerate while ketoisoleucine resulted mainly in release of lactate and ketone bodies. Accumulation of metabolites from the branched-chain amino acids is discussed in relation to the metabolism of the amino acids in skeletal muscle and liver of rat.

3-Hydroxybutyric Acid↗
PubMed1992

A site-directed mutagenesis study on the role of isoleucine-23 of human epidermal growth factor in the receptor binding.

The isoleucine-23 residue of human epidermal growth factor (hEGF) was substituted by a variety of amino acid residues and the receptor-binding activities of variant hEGFs were determined by the use of human KB cell. Tight receptor binding was found of variants with hydrophobic amino acid residues in position 23. The size of the isoleucine residue was nearly optimum for the receptor binding as compared with other hydrophobic residues. The structure analysis by two-dimensional nuclear magnetic resonance spectroscopy showed that the substitution at position 23 only slightly affected the tertiary structure of hEGF. These indicate that the side chain of isoleucine residue in position 23, which is exposed on the protein surface, directly binds to a hydrophobic pocket of the receptor.

Amino Acid Sequence↗
PubMed2005

Lack of carcinogenicity of L-isoleucine in F344/DuCrj rats.

The carcinogenic potential of L-isoleucine, used as a food fortifier, was examined in both sexes of F344 rats. Groups of 50 female and 50 male animals were given diet containing L-isoleucine at concentrations of 0%, 2.5% or 5.0%. No treatment-related changes in the survival rate, general condition, body weight, food consumption, urinalysis, hematology or clinical chemistry data and organ weights were noted. Detailed histopathological examination revealed no treatment-related increase in the incidences of any non-neoplastic or neoplastic lesions. The results indicate that L-isoleucine is not carcinogenic in F344 rats of either sex.

Animals↗
PubMed1998

Ethylmalonic and methylsuccinic aciduria in ethylmalonic encephalopathy arise from abnormal isoleucine metabolism.

Ethylmalonic encephalopathy (EE), an organic aciduria of unknown etiology characterized by developmental delay, hypotonia, and vascular instability associated with lactic acidemia and urinary excretion of ethylmalonic acid (EMA) and methylsuccinic acid (MSA), has been described in 11 patients. To test the possibility that the underlying biochemical defect involves isoleucine catabolism, we determined the response to oral L-isoleucine (IIe) load (150 mg/kg) in a 5-year-old girl with EE and in three healthy, age- and sex-matched controls. Following IIe load in the patient, there was accumulation of 2-methylbutyrylglycine (2-MBG) and a delayed and lower peak urinary excretion of tiglylglycine (TGL), suggesting a partial defect in 2-methyl-branched chain acylcoenzyme A dehydrogenase (2M-BCAD). In vitro measurements 2M-BCAD activity in cultured skin fibroblasts from patients with EE have been reported to be normal. Our results show that isoleucine is a source for the elevated EMA and MSA in patients with EE, and suggest a functional, possibly secondary, deficiency of activity of 2M-BCAD in vivo.

Administration, Oral↗
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↗
PubMed1992

The anti-tumour effects of the prodrugs N-l-leucyl-doxorubicin and vinblastine-isoleucinate in human ovarian cancer xenografts.

N-l-leucyl-doxorubicin and vinblastine-isoleucinate can be considered as relatively non-toxic prodrugs from doxorubicin and vinblastine, respectively. A comparative analysis was carried out of the anti-tumour activity of the four compounds as well as vintriptol in four human ovarian cancer xenografts different in histology, growth rate and chemosensitivity. Injections were given i.v. weekly twice into mice bearing well-established s.c. tumours. At equitoxic doses, the amount of drug administered for N-l-leucyl-doxorubicin and vinblastine-isoleucinate was respectively 3-fold and 2-fold higher than the doses of the parent compound. N-l-leucyl-doxorubicin induced a growth inhibition > 50% in three out of four human ovarian cancer lines. The anti-tumour effects obtained were significantly better (P < 0.01) than in the case of doxorubicin. Vinblastine-isoleucinate studied in two of these lines could induce a growth inhibition of > 50%. This prodrug appeared slightly less effective than vinblastine. Insignificant growth inhibition (< 50%) was obtained by vintriptol.

Animals↗
PubMed1976

Demonstration of a new mammalian isoleucine catabolic pathway yielding an Rseries of metabolites.

1. Normal human urine contains small amounts (less than 4 mg/g of creatinine) of 2-ethylhydracrylic acid, formed, we believe, by a previously undisclosed endogenous catabolic pathway for the oxidation of a newly described series of R metabolites of isoleucine. 2. Urinary excretion of 2-ethylhydracrylic acid is variably increased in defects of isoleucine oxidation at distal steps in the catabolic pathway (3-oxoacyl-CoA thiolase deficiency and methylmalonyl-CoA mutase deficiency) and is diminished when proximal steps of the oxidative pathway are blocked as in branched-chain oxo acid decarboxylase deficiency ('maple-syrup-urine' disease). 3. Precursors of R-pathway metabolites [R(-)-2-methylbutyrate and 2-ethylacrylate ] lead to increased 2-ethylhydracrylate excretion in the mammal(rat, rabbit and dog); the corresponding S metabolites [S(+)-2-methylbutyric acid and tiglic acid ], when given in equimolar amounts, have little effect on its excretion, suggesting that little or no interconversion between S and R metabolites occurs in vivo. 4. Studies with 2H-labelled precursors indicate that conversion of R 2-methylbutyrate into 2-ethylhydracrylic acid occurs by a direct pathway (apparently via 2-ethylacrylic acid). 5. The further oxidation of 2-ethylhydracrylic acid to ethylmalonic acid was demonstrated, and may be analogous to S-metabolite oxidation via methyl malonate. 6. Valine metabolites do not interact with the R=isoleucine pathway under the conditions of these experiments in vivo.

Acrylates↗
PubMed1994

Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c.

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

Animals↗
PubMed1999

Upper gastrointestinal bleeding: an ammoniagenic and catabolic event due to the total absence of isoleucine in the haemoglobin molecule.

Upper gastrointestinal bleeding causes increased urea concentrations in patients with normal liver function and high ammonia concentrations in patients with impaired liver function. This ammoniagenesis may precipitate encephalopathy. The haemoglobin molecule is unique because it lacks the essential amino acid isoleucine and has high amounts of leucine and valine. Upper gastrointestinal bleeding therefore presents the gut with protein of very low biologic value, which may be the stimulus to induce a cascade of events culminating in net catabolism. This may influence the function of rapidly dividing cells and short half-life proteins. We hypothesize that, following a variceal bleed in a cirrhotic patient, the lack of isoleucine in blood protein is the cause of the exaggerated ammoniagenesis and catabolism. We propose that intravenous administration of isoleucine may serve as a simple therapeutic that transforms blood protein in a balanced protein, resulting in only a short-lived rise in ammonia and urea production, and preventing interference with protein synthesis.

Ammonia↗
PubMed1971

Involvement of threonine deaminase in multivalent repression of the isoleucine-valine pathway in Saccharomyces cerevisiae.

A strain (MAR33) of Saccharomyces cerevisiae containing a threonine deaminase [L-threonine hydrolyase (deaminating) EC 4.2.1.16] with decreased feedback sensitivity has been shown to have a specific activity of acetohydroxy acid synthetase higher than that of the parent strain (MD11) when both are grown on minimal medium. When strain MAR33 is grown on minimal medium supplemented only with isoleucine, the specific activity of the synthetase is reduced to that found in the parent strain. Another strain, D106-1A, contains a nonsense mutation in the middle of the gene for threonine deaminase. When this strain is grown on minimal medium containing appropriate supplements (which include a nonrepressing concentration of isoleucine), or on minimal medium supplemented with isoleucylglycine (which acts as a limiting source of isoleucine), acetohydroxy acid synthetase remains repressed. Leucine limitation causes partial derepression. With the reversion of the nonsense mutation, either intragenically or via a suppressor for the mutation, partial derepression of the synthetase returns. When D106-1A is diploidized with either M15, a mutant lacking the synthetase, or MD9, a strain containing the enzyme, normal, partially derepressed, values for this enzyme are found. This indicates that threonine deaminase is necessary for derepression, and that it possibly acts as an inducer.

Culture Media↗
PubMed2003

Cell surface expression of the melanocortin-4 receptor is dependent on a C-terminal di-isoleucine sequence at codons 316/317.

Loss-of-function mutations in the human melanocortin-4 receptor (MC4R) are associated with obesity. Previous work has implicated a C-terminal di-isoleucine motif at residues 316/317 in MC4R cell surface targeting. It was therefore of interest to examine function and cell surface expression of an MC4R mutation found in an obese proband in which one of these isoleucines was substituted by threonine (I317T). Single mutant (I316T or I317T) and double mutant (I316T,I317T) forms of MC4R were constructed by oligonucleotide-directed mutagenesis and tested for function and cell surface expression in transfected cells. Function was assessed using assays for agonist, [Nle(4)-d-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) or forskolin-stimulated cAMP accumulation. Cell surface expression was determined by whole-cell binding of [(125)I]NDP-alpha-MSH, fluorescence immunocytochemistry and fluorescence-activated cell sorting. Maximal cAMP generation of the single mutants was reduced by 40% of wild-type receptor; the double mutant further reduced function to 40% of control, effects that were mirrored by decreases in cell-surface expression. Quantitative RT-PCR showed that, relative to wild-type receptor, transcript levels for the mutated receptors were not reduced. The results further implicate the C-terminal di-isoleucines in cell surface expression of MC4R and suggest that mutations of residues 316 or 317 would predict MC4R hypofunction.

Amino Acid Sequence↗
PubMed1976

Amino acid metabolism in the piglet. 2. Influence of fasting on plasma free amino acid concentration and in vivo oxidation of methionine, isoleucine and threonine.

1. The influence of a 24 h fast on the concentrations of free amino acids in the plasma, and upon the oxidation rates of methionine, isoleucine and threonine was studied (using early weaned, 4-week-old piglets which were receiving a semi-purified diet. 2. There was no change in the total concentration of the essential amino acids as a result of the 24 h fast: the concentration of the branched-chain amino acids increased, but the effect of this was offset by decreases in the concentrations of arginine, histidine, lysine, methionine and phenylalanine. There was a reduction in the concentration of the non-essential amino acids. 3. The piglets received infusions of L-[I-14C]methionine, L-[U-14C]isoleucine and L-[U-14C]-threonine, and the recovery of the label in carbon dioxide was determined. Less than 5% of the activity from methionine was recovered in the CO2 from the fed piglets, whereas 12% was recovered from the fasted piglets. The corresponding values with threonine were 11 and 19% but there was no effect of fasting on the recovery of the label from isoleucine in CO2. 4. The initial dilution of a single dose of a labelled amino acid infused into the bloodstream depends on the plasma concentration of the amino acid. Nutritional regimens may effect the free amino acid concentration in the plasma. Thus comparisons based upon direct determination of activity recovered in CO2 from the labelled dose of an amino acid with animals on different nutritional regimens could not misleading, unless the differences in the concentrations of the amino acid in the plasma are considered.

Amino Acids↗