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At least 109 records · Page 6Linked to original sources
PubMed1998

Assessment of whole body L-leucine oxidation by noninvasive L-[1-13C]leucine breath tests: a reappraisal in patients with maple syrup urine disease, obligate heterozygotes, and healthy subjects.

Suitability of a recently proposed noninvasive L-[13C]leucine breath test for assessment of whole body leucine oxidation in maple syrup urine disease (MSUD) was examined. Oral L-[1-13C]leucine loads (38 micromol/kg body weight) were performed in overnight fasted MSUD patients (n = 6, classical form), obligate heterozygote parents (n = 6), and control subjects (n = 10). Three-hour 13CO2 exhalation kinetics were evaluated using curve fitting procedures. Venous blood was obtained in most cases and analyzed for 13C-labeled plasma metabolites. In control subjects, maximal 13CO2 exhalation was reached at tmax = 55 +/- 18 min. Cumulative 13CO2 output at 3 h amounted to 4.7 +/- 0.7 micromol x (kg body weight)(-1). Estimated total 3CO2 exhalation was 7.2 +/- 1.4 micromol x (kg body weight)(-1) (19.0 +/- 3.6% of the dose). Half of this amount was expired at t1/2 = 130 +/- 18 min. The data show a considerable degree of intersubject variability. Intraindividual variability was comparable, however, when checked in two volunteers. In obligate heterozygotes, 13CO2 kinetics were similar to controls (tmax = 35 +/- 8 min, t1/2 = 95 +/- 16 min). Total 13CO2 output [5.7 +/- 1.4 micromol x (kg body weight)(-1)] tended to be in the lower control range. None of the MSUD patients under study exhibited a significant increase in 13CO2 output after load. Maximal increase of label in plasma 4-methyl-2-oxopentanoate, the physiologic precursor of 13CO2, was 16.1 +/- 3.5 MPE in control subjects. In MSUD, label dilution was increased and correlated with the patients' leucine/4-methyl-2-oxopentanoate plasma levels. Considering the generally high variability of 13CO2 output and the unstable substrate pools in MSUD, we discuss the limitations of whole body leucine oxidation measurements by noninvasive approaches.

Adolescent↗
PubMed1976

Leucine 2,3-aminomutase, an enzyme of leucine catabolism.

The initial step in the fermentation of leucine to acetate, isobutyrate, and ammonia by Clostridium sporogenes is the B12 coenzyme-dependent conversion of alpha-leucine to beta-leucine (3-amino-4-methylpentanoate). The amino group migration reaction, catalyzed by leucine 2,3-aminomutase, is reversible and is inhibited by intrinsic factor. The enzyme activity has been found in several clostridia, in rat, sheep, rhesus, and African green monkey livers, and in human leukocytes.

Amino Acid Isomerases↗
PubMed1994

Formation of leucyl-leucine-O-methylester in leucine-O-methylester treated cells.

Porcine polymorphonuclear cells (PMN) and murine macrophages (M phi) were treated in vitro with Leu-OMe or Leu-Leu-OMe (1.5-5.0 mM) for various periods of time. It was found that the Leu-OMe and Leu-Leu-OMe entered cells rapidly, concomitantly the intracellular leucine accumulated. The methyl derivative diffused faster than Leucine due to its lipophylic character. The Leucine-O-methylesters hydrolysed rapidly as a consequence of the esterase and peptidase activities. The cells treated with Leu-OMe accumulated a high amount of Leucine and some Leu-Leu-OMe too. It was found that the formation of the didpeptide-methylester is not a spontaneous process, rather an enzymatic one. The Leu-OMe treated cells serve as a model which can be used to investigate the effect of the amino acid metabolism and the formation of dipeptides intracellularly and extracellularly.

Animals↗
PubMed1999

Molecular cloning of adipocyte-derived leucine aminopeptidase highly related to placental leucine aminopeptidase/oxytocinase.

In the current study, we report the cloning and initial characterization of a novel human cytosolic aminopeptidase named adipocyte-derived leucine aminopeptidase (A-LAP). The sequence encodes a 941-amino acid protein with significant homology (43%) to placental leucine aminopeptidase (P-LAP)/oxytocinase. The predicted A-LAP contains the HEXXH(X)18E consensus sequence, which is characteristic of the M1 family of zinc-metallopeptidases. Although the deduced sequence contains a hydrophobic region near the N-terminus, the enzyme localized mainly in cytoplasm when expressed in COS-7 cells. Northern blot analysis revealed that A-LAP was expressed in all the tissues tested, some of which expressed at least three forms of mRNA, suggesting that the regulation of the gene expression is complex. When aminopeptidase activity of A-LAP was measured with various synthetic substrates, the enzyme revealed a preference for leucine, establishing that A-LAP is a novel leucine aminopeptidase with restricted substrate specificity. The identification of A-LAP, which reveals strong homology to P-LAP, might lead to the definition of a new subfamily of zinc-containing aminopeptidases belonging to the M1 family of metallopeptidases.

Adipocytes↗
PubMed1994

Nitrogen homoeostasis in man: diurnal changes in nitrogen excretion, leucine oxidation and whole body leucine kinetics during a reduction from a high to a moderate protein intake.

1. The adaptation of the diurnal cycle of nitrogen (N) homeostasis during a change in protein intake was investigated with diurnal measurements of N and leucine balance and turnover during a reduction from a high to a moderate protein intake in normal adults. 2. In experiment 1, during a 9 day period after a reduction from 1.82 to 0.77 g of protein day-1 kg-1, N excretion fell slowly at a similar rate in fed and fasted states so that the lowered intake was unable to replete any of the postabsorptive losses for 3 days. There was a marked negative N balance, which persisted throughout the study, although with a significant reduction in N losses in both fed and fasted states on day 4, balances during days 4-9 (-32.8 +/- 28.3 mg of N day-1 kg-1) were less negative than during days 1-3 (-79.1 +/- 60.4 mg of N day-1 kg-1). 3. In experiment 2, during a 14 day period after a reduction from 1.89 to 0.77 g of protein day-1 kg-1, [1-13C]leucine oxidation and turnover were measured by primed intravenous infusion, during fasting and feeding in subjects before and on days 3, 7 and 14 after the dietary change. Leucine oxidation fell by 32% (P < 0.05) on day 3 in the fed state and by 12% (P < 0.05) during fasting, falling further in each case by day 7 with improved balance.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
PubMed2001

Inhibition of beta(2) integrin-mediated leukocyte cell adhesion by leucine-leucine-glycine motif-containing peptides.

Many integrins mediate cell attachment to the extracellular matrix by recognizing short tripeptide sequences such as arginine-glycine-aspartic acid and leucine-aspartate-valine. Using phage display, we have now found that the leukocyte-specific beta(2) integrins bind sequences containing a leucine-leucine-glycine (LLG) tripeptide motif. An LLG motif is present on intercellular adhesion molecule (ICAM)-1, the major beta(2) integrin ligand, but also on several matrix proteins, including von Willebrand factor. We developed a novel beta(2) integrin antagonist peptide CPCFLLGCC (called LLG-C4), the structure of which was determined by nuclear magnetic resonance. The LLG-C4 peptide inhibited leukocyte adhesion to ICAM-1, and, interestingly, also to von Willebrand factor. When immobilized on plastic, the LLG-C4 sequence supported the beta(2) integrin-mediated leukocyte adhesion, but not beta(1) or beta(3) integrin-mediated cell adhesion. These results suggest that LLG sequences exposed on ICAM-1 and on von Willebrand factor at sites of vascular injury play a role in the binding of leukocytes, and LLG-C4 and peptidomimetics derived from it could provide a therapeutic approach to inflammatory reactions.

Amino Acid Motifs↗
PubMed2001

Isolation of homeodomain-leucine zipper genes from the moss Physcomitrella patens and the evolution of homeodomain-leucine zipper genes in land plants.

Homeobox genes encode transcription factors involved in many aspects of developmental processes. The homeodomain-leucine zipper (HD-Zip) genes, which are characterized by the presence of both a homeodomain and a leucine zipper motif, form a clade within the homeobox superfamily and were previously reported only from vascular plants. Here we report the isolation of 10 HD-Zip genes (named PPHB:1-PPHB:10) from the moss Physcomitrella patens. Based on a phylogenetic analysis of the 10 PPHB: genes and previously reported vascular plant HD-Zip genes, all of the PPHB: genes except Pphb3 belong to three of the four HD-Zip subfamilies (HD-Zip I, II, and III), indicating that these subfamilies originated before the divergence of the vascular plant and moss lineages. Pphb3 is sister to the HD-Zip II subfamily and has some distinctive characteristics, including the difference of the a(1) and d(1) sites of its leucine zipper motif, which are well conserved in each HD-Zip subfamily. Comparison of the genetic divergence of representative HD-Zip I and II genes showed that the evolutionary rate of HD-Zip I genes was faster than that of HD-Zip II genes.

Amino Acid Sequence↗
PubMed1999

Characterization of homeodomain-leucine zipper genes in the fern Ceratopteris richardii and the evolution of the homeodomain-leucine zipper gene family in vascular plants.

The homeodomain-leucine zipper (HD-Zip) genes encode transcription factors that are characterized by the presence of both a homeodomain and a leucine zipper motif. They belong to the homeobox gene superfamily and have been reported only from flowering plants. This article is the first report on the ferm HD-Zip genes (named Crhb1-Crhb11) isolated from the homosporous ferm Ceratopteris richardii. Phylogenetic analyses of the II Crhb genes with previously reported angiosperm HD-Zip genes show that the Crhb genes belong to three of the four different angiosperm HD-Zip subfamilies (HD-Zip I, II, and IV), indicating that these subfamilies of HD-Zip genes originated before the diversification of the ferm and seed plant lineages. The Crhb4-Crhb8 and Crhb11 genes belong to the HD-Zip I subfamily but differ from angiosperm HD-Zip I genes by the presence of a seven-amino-acid indel in the leucine zipper motif. By the northern analyses, Crhb1 and Crhb3 were expressed only in gametophyte tissue. Expression of Crhb2 and Crhb11 genes could not be detected in any tissue examined, while all other Crhb genes were expressed in most sporophytic and gametophytic tissues. Although the functions of the Crhb genes in Ceratopteris are unknown, their patterns of expression suggest that they regulate developmental or physiological processes common to both the gametophyte and the sporophyte generations of the fern. Differences in the expression of Crhb1 between male gametophytes and male-hermaphrodite mixed populations of gametophytes suggests that the Crhb1 gene is involved in gametophytic sex determination.

Amino Acid Sequence↗
PubMed1981

[Physiological importance of microvilli-bound leucine arylamidase in the final digestion of proteins. 1. Purification and isolation of intestinal leucine arylamidase and aminotripeptidase of rats].

The membrane-bound leucine arylamidase of the microvilli of the rat small intestine was solubilized by Triton X-100 and purified by gel and ion-exchange chromatography. As compared to the mucosa homogenate, the purification factor was 135. The leucine arylamidase and aminotripeptidase of the microvilli cannot be separated by chromatography. The cytosomal portion of the aminopeptidase is devoid of leucine arylamidase activity.

Aminopeptidases↗
PubMedDate not supplied

Blood pressure responses of conscious rats to intravenous administration of enkephalin derivatives (D-ala2 methionine and leucine enkephalinamide, and methionine and leucine enkephalinamide.

The present study was designed to determine the blood pressure (BP) responses of conscious rats given intravenous (IV) injections of enkephalin derivatives (D-ala2-methionine enkephalinamide, DAMEA; D-ala2-leucine enkephalinamide, DALEA; methionine enkephalinamide, MEA; leucine enkephalinamide, LEA) and the receptor mechanisms mediating the resultant change in BP. IV injection of 1.6-16.0 nmoles of DAMEA or DALEA caused a transient but potent decrease in mean arterial pressure (MAP) and mean heart rate (MHR). LEA and MEA (16.0 nmoles) given IV produced slight pressor responses, which were not associated with concomitant tachycardia whereas 48 nmoles of MEA elicited a hypotensive effect accompanied by a fall in MHR. Pretreatment studies whereby various receptor antagonists (naloxone, diprenorphine, phentolamine, D-L-propranolol or atropine) were given IV 5 min before subsequent IV administration of DAMEA, DALEA, MEA or LEA (16 nmoles) showed that naloxone, diprenorphine and atropine blocked the depressor and bradycardic effects of DALEA and DAMEA. Naloxone and phentolamine suppressed the pressor response of both MEA and LEA (16.0 nmoles) while diprenorphine blocked the rise in MAP to only MEA. The results show that DAMEA and DALEA mediate their depressor actions in conscious rats via a negative chronotropic effect through an interaction of muscarinic cholinergic receptors on the myocardium. It suggested that the pressor response of MEA and LEA may be produced via an alpha-receptor mediated effect on the peripheral vasculature to cause vasoconstriction.

Analgesics↗
PubMed2000

Effect of lengthening of peptide backbone by insertion of chiral beta-homo amino acid residues: conformational behavior of linear peptides containing alternating L-leucine and beta-homo L-leucine residues.

The synthesis and the solution behavior of the linear peptides containing a beta-homo (beta-H) leucine residue-Boc-Leu-beta-HLeu-Leu-OMe, Boc-beta-HLeu-Leu-beta-HLeu-Leu-OMe, and Boc-Leu-beta-HLeu-Leu-beta-HLeu-Leu-OMe-as well as the solid structure of the tripeptide, are reported. The conformational behavior of the peptides was investigated in solution by two-dimensional nmr. Our data support the existence in solution with different families of conformers in rapid interchange. The crystals of the tripeptide are orthorhombic, space group P2(1)2(1)2, with a = 15.829(1) A, b = 29.659(1) A, c = 6.563(1) A, and Z = 4. The structure has been solved by direct methods and refined to final R1 and wR2 indexes of 0.0530 and 0.1436 for 2420 reflections with I > 2sigma(I). In the solid state, the tripeptide does not present intramolecular H bonds, and the peptide backbone of the two leucine residues adopts a quasi-extended conformation. For the beta-HLeu residue, the backbone conformation is specified by the torsion angles straight phi(2) = -120.9(4) degrees, mu(2) = 56.7(4) degrees, psi(3) = -133.2(4) degrees. The side chains of the three residues assume the same conformation (g(-), g(-), trans), and all peptide bonds, except the urethane group at the N-terminus, are in the trans conformation. Preliminary conformational energy calculations carried out on the Ac-NH-beta-HAla-NHMe underline that the conformations with mu angle equal to 180 degrees and 60 degrees assume lower energy with respect to the others. In addition, we found a larger conformational freedom for the psi angle with respect to the straight phi angle.

Crystallography, X-Ray↗
PubMed1993

Human peripheral blood gamma delta T cells are uniformly sensitive to destruction by the lysosomotropic agents leucine methyl ester and leucyl leucine methyl ester.

Treatment of peripheral blood mononuclear cells (PBMC) with the lysosomotropic agent leucine methyl ester (Leu-OMe) eliminates monocytes/macrophages and cytotoxic lymphocytes including CD3- CD16+ natural killer (NK) cells and a fraction of T cell receptor (TcR) alpha beta + CD8+ T cells. We report that freshly isolated peripheral blood gamma delta T cells are highly sensitive to Leu-OMe treatment as well. After incubation of PBMC with 5 mM Leu-OMe or incubation of purified T cells with 50 microM leucyl leucine methyl ester (Leu-Leu-OMe) and subsequent overnight culture, CD3-CD16+ NK cells and gamma delta T cells were no longer detectable by immunofluorescence analysis. The two major gamma delta T cell subsets V gamma 9+V delta 2+ and V gamma 9-V delta 1+ were equally susceptible to Leu-OMe and Leu-Leu-OMe treatment. The elimination of V gamma 9+ T cells by Leu-OMe treatment was confirmed in functional assays. Stimulation of peripheral blood T cells with killed mycobacteria resulted in selective expansion of V gamma 9+ T cells. In contrast, no activation of gamma delta T cells was elicited in Leu-OMe-treated responder T cells stimulated with killed mycobacteria.

Blood↗
PubMed1994

Simultaneous determination of plasma levels of alpha-ketoisocaproic acid and leucine and evaluation of alpha-[1-13C]ketoisocaproic acid and [1-13C]leucine enrichment by gas chromatography-mass spectrometry.

A sensitive method for the simultaneous analysis of plasma alpha-ketoisocaproic acid (KIC) and leucine using GC-MS is described. Plasma was deproteinized after addition of both alpha-ketovaleric acid and norleucine as internal standards. Derivatization of the compounds involved protection of the keto groups with methoxyamine and tert-butyldimethylsilylation under optimized conditions. Mass spectral analyses were carried out in electron impact ionization mode and the gas chromatographic conditions selected prevented interference by other keto acids and amino acids normally present in plasma or by the biological matrix. Determination of both plasma levels and isotopic enrichment of KIC and leucine required a single injection and was easily accomplished in 7-9 min. The good reproducibility of the method, in addition to rapidity and simplicity, permits several samples to be accurately analyzed daily. Therefore, the method is particularly useful for studies on the metabolism of amino acids.

Carbon Isotopes↗
PubMed2004

Generating oxidation-resistant variants of Bacillus kaustophilus leucine aminopeptidase by substitution of the critical methionine residues with leucine.

Bacillus kaustophilus leucine aminopeptidase (bkLAP) was sensitive to oxidative damage by hydrogen peroxide. To improve its oxidative stability, the oxidation-sensitive methionine residues in the enzyme were replaced with leucine by site-directed mutagenesis. The variants, each with an apparent molecular mass of approximately 54 kDa, were overexpressed in recombinant Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. The specific activity for M282L, M285L, M289L and M321L decreased by more than 43%, while M400L, M426L, M445L, and M485L showed 191, 79, 313, and 103%, respectively, higher activity than the wild-type enzyme. Although the mutations did not cause significant changes in the K(m) value, more than 67.8% increase in the value of k(cat)/K(m) was observed in the M400L, M426L, M445L and M485L. In the presence of 50 mM H2O2, most variants were more stable with respect to the wild-type enzyme, indicating that the oxidative stability of the enzyme can be improved by engineering the methionine residues.

Amino Acid Substitution↗
PubMed1985

Ethanol and leucine oxidation--II. Leucine oxidation by rat tissue in vitro.

The effect of ethanol upon leucine oxidation by rat tissues in vitro is reported. The activities of branched chain amino acid aminotransferase and 2-oxo acid dehydrogenase were decreased by chronic administration of ethanol (20% v/v solution as drinking water for 35 d) in muscle and kidney but were increased, although not significantly, in liver. Acute administration of ethanol (8 g kg-1 body-weight 0.73) did not affect enzyme activities. Tissue NAD+:NADH ratios, calculated from lactate:pyruvate ratios, were significantly decreased in the liver and kidney of rats receiving ethanol acutely. These data are consistent with the view that ethanol decreases leucine oxidation by decreasing availability of NAD+ when given acutely and by decreasing enzyme activity when administered chronically.

Animals↗
PubMed1987

Metabolism of leucine in fibroblasts with defects in the leucine catabolic pathway.

1. The activity of the leucine catabolic pathway was measured by quantitation of the radiolabeled glutamic acid produced by intact cultured fibroblasts incubated with radiolabeled leucine. 2. Five normal cell lines produced an average of 179 +/- 47 pmol glutamic acid/mg cell protein/hr. 3. Six cell lines with defects in one of the enzymes of the pathway showed activities of 0-40 pmol glutamic acid/mg cell protein/hr. 4. Each of the cell lines with an enzyme defect produced a characteristic pattern of radiolabeled organic acids. In every case, the amounts of these organic acids produced was less than the amount of glutamic acid produced by normal cells. None of the cell lines accumulated CoA esters.

Amino Acid Metabolism, Inborn Errors↗
PubMed2000

Possibility of wound dressing using poly(L-leucine)/poly(ethylene glycol)/poly(L-leucine) triblock copolymer.

ABA-type block copolymers (abbreviated as LEL) composed of poly(L-leucine) (PLL) as the A component and poly(ethylene glycol) (PEG) as the B component were synthesized by ring-opening polymerization of L-leucine N-carboxyanhydride initiated by primary amino group located at both ends of PEG chain. A silver sulfadiazine (AgSD)-impregnated wound dressing of sponge type was prepared by the lyophilization method. Morphological structure of this wound dressing by scanning electron microscopy was observed to be composed of a dense skin layer and a porous inner layer. Equilibrium water content of LEL wound dressing increased with an increase in PEG content in the block copolymer due to the hydrophilicity of PEG. AgSD release from AgSD-impregnated wound dressing in PBS buffer (pH = 7.4) was dependent on PEG content in the block copolymer. Release of AgSD was increased in proportion to the PEG content in the copolymer. Antibacterial capacity of AgSD-impregnated wound dressing was examined in agar plate against Pseudomonas aeruginosa and Staphylococcus aureus. It was found that the suppression of bacterial proliferation in the wound dressing was dependent upon the PEG content. In cytotoxicity test, cell damage did not occur by the release of AgSD from the LEL sponge matrix of AgSD-medicated wound dressing. In in vivo test, granulous tissue formation and wound contraction for the AgSD- and dehydroepiandrosterone-impregnated LEL-2 wound dressing were faster than for any other groups.

Animals↗
PubMed2000

Insulin reduces leucine oxidation and improves net leucine retention in parenterally fed humans.

Protein metabolism during parenteral feeding was measured with and without euglycemic hyperinsulinemic clamping in five healthy human subjects using the primed continuous infusion of [(13)C]leucine (LEU) tracer methodology. All subjects underwent two periods of protein measurement. Subjects were randomized to have the clamp first or second. Two subjects had the clamp first, and they participated in another study in which the measurement period after the clamp was increased by another 2 h. Insulin reduced LEU oxidation (from 26 to 22 microM. kg(-1). h(-1); P < 0.05) and improved net LEU balance (from 3 to 7 microM. kg(-1). h(-1); P < 0. 05). The order of the clamp influenced the effects of insulin. With clamping performed second, insulin reduced LEU flux (from 152 to 134 microM. kg(-1). h(-1); P < 0.01), endogenous LEU rate of appearance (Ra; from 125 to 107 microM. kg(-1). h(-1); P < 0.01), and non-oxidative LEU disappearance (NOLD; from 128 to 113 microM. kg(-1). h(-1); P < 0.01). With clamping performed first, NOLD decreased after insulin was stopped (from 129 to 121 microM. kg(-1). h(-1); P < 0.05), but no change was seen in the flux and LEU Ra, despite the return of plasma insulin and amino acid concentrations to basal levels. The reduction in NOLD was accentuated with time and did not reach plateau even after 6 h and indicated a prolonged carry-over effect for NOLD and Ra. This effect was not seen for leucine oxidation.

Adult↗