PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “LEUCINE AMINOPEPTIDASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed1990

Schistosoma mansoni: purification and characterization of a membrane-associated leucine aminopeptidase.

Membrane-associated leucine aminopeptidase (EC 3.4.11.1, LAP) has been purified to homogeneity from Schistosoma mansoni egg homogenates by a combination of ultracentrifugation, chromatofocusing, and molecular sieve chromatography. A 260-fold increase in specific activity was observed after purification. This is a metalloenzyme, containing carbohydrate moieties. Optimal enzyme activity was found at neutral pH. Enzyme activity was measured using L-leucine-7-amino-4-trifluoromethylcoumarin (L-Leu-AFC); in addition, schistosome egg LAP hydrolyzed a variety of other aminopeptidase substrates. Hydrolysis of L-Leu-AFC was inhibited by a number of aminopeptidase inhibitors, including 1,10-phenanthroline, bestatin, and amastatin.

Animals↗
PubMed1986

Immunoaffinity purification and characterization of leucine aminopeptidase from human liver.

Leucine aminopeptidase was purified from human liver cytosol to homogeneity, 1538-fold, with a yield of 84.4% by immunoaffinity chromatography. Increases in the activity and the stability of the enzyme were simultaneously observed during the purification procedure, suggesting the presence of some endogenous inhibitor in cytosol. The specific activity and Km value of the enzyme for L-leucine amide were found to be 58.00 mumol/min/mg of protein and 4.02 mM, respectively, at pH 8.0. The molecular weight of the enzyme was determined to be 360,000 by both polyacrylamide gradient gel electrophoresis and Sephadex G-200 gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of native and dimethyl suberimidate cross-linked enzyme indicate that the native enzyme has two subunits of Mr 53,000 (a) and 65,000 (b) and is a hexamer arranged as a trimer of dimers (3 X (a X b)). The optimum pH was 10.5, and the enzyme was stable in the pH range from 7.5-8.5. The enzyme was activated by divalent metal ions, especially by Mg2+ and Mn2+, with no change in Km value. The enzyme was inhibited by metal-chelating agents, indicating it to be a metalloenzyme. Amastatin and bestatin strongly inhibited the enzyme, but leupeptin did not. The enzyme had a broad substrate specificity toward oligopeptides and amino acid amides but had little or no activity toward chromogenic substrates. The enzyme also could hydrolyze natural substrates contained in liver cytosol and accordingly produce many kinds of amino acids commonly found in proteins.

Cations, Divalent↗
PubMed1979

[Serum activity of leucine aminopeptidase in lymphotropic infections and in malignant lymphomas].

Increases of the leucine aminopeptidase according to data of literature are regarded as a sensitive parameter of lesions in liver diseases. In patients with active generalised lymphadenitis (viral infections, toxoplasmosis) as a typical enzyme constellation a relatively strong leucine aminopeptidase increase in the serum with missing or relatively slight enzyme deviation of the transaminases could be found. The quotient of the activities of LAP/ALAT was clearly above that of inflammatory liver diseases. In chronic lymphatic leukosis, plasmocytoma and malignant lymphomas the leucine aminopeptidase serum activities were within the normal. Increases of leucine aminopeptidase in lymphotropic infections are probably partly of extrahepatic origin.

Humans↗
PubMed1993

Characterization of the Rickettsia prowazekii pepA gene encoding leucine aminopeptidase.

The pepA gene, encoding a protein with leucine aminopeptidase activity, was isolated from Rickettsia prowazekii, an obligate intracellular parasitic bacterium. Nucleotide sequence analysis revealed an open reading frame of 1,502 bp that would encode a protein of 499 amino acids with a calculated molecular weight of 53,892, a size comparable to that of the protein produced in Escherichia coli minicells containing the rickettsial gene. Also, heat-stable leucine aminopeptidase activity was demonstrable in an E. coli peptidase-deficient strain containing R. prowazekii pepA. Comparison of the amino acid sequence of the R. prowazekii PepA with the characterized leucine aminopeptidases from E. coli, Arabidopsis thaliana, and bovine eye lens revealed that 39.8, 34.9, and 34.0% of the residues were identical, respectively. Residues proposed to be part of the active site or involved in the binding of metal ions in the bovine metalloenzyme were all conserved in R. prowazekii PepA. However, despite the structural and enzymatic similarity to E. coli PepA, the R. prowazekii protein was unable to complement the cer site-specific, PepA-dependent recombination system found in E. coli that resolves ColE1-type plasmid multimers into their monomeric forms.

Amino Acid Sequence↗
PubMed1983

Kinetic parameters of metal-substituted leucine aminopeptidase from bovine lens.

Leucine aminopeptidase (LAP) is a protease requiring two divalent metal cations per subunit for activity. Zn2+, Mg2+, and Co2+ metal-substituted forms of LAP have been prepared and investigated kinetically. Substitution of metal into the two binding sites independently resulted in the preparation of Zn2+Zn2+, Mg2+Zn2+, Co2+Co2+, Zn2+Co2+, Mg2+Co2+, and Co2+Zn2+ LAP derivatives that were characterized by atomic absorption spectrophotometry. Kinetic analysis of the metal-substituted enzymes indicated that site 1 (fast exchanging) metal substitution results in a Km decrease in the relative order Zn2+ greater than Mg2+ greater than Co2+. Similar comparisons for the site 2 metal (slow exchanging) involved only Zn2+ and Co2+, since only these metals have been shown to compete effectively for this site. Substitution of these two metals into site 2 revealed a Km decrease in the order Zn2+ greater than Co2+. It was suggested previously [e.g., Thompson, G. A., & Carpenter, F. H. (1976) J. Biol. Chem. 251, 1618-1624] that the fast-exchanging site 1 metal predominantly effects kcat while the slow-exchanging metal in site 2 exerts effects exclusively on Km. The present study, the first direct comparison of Km change resulting from metal substitution into both sites, clearly indicates that both metal sites exert significant effects on Km. In addition, the data suggest a more complex interaction between the two bound metals than previously suspected.

Animals↗
PubMed1996

Localization and post-translational processing of the wound-induced leucine aminopeptidase proteins of tomato.

Leucine aminopeptidase (LAP) is induced by wounding and bacterial pathogen infection in tomato. DNA blot analysis of XbaI-digested lambdalap genomic clones demonstrated that LapA1 and LapA2 cDNAs were encoded by two different LapA genes in the tomato genome. The coding and untranslated regions of LapA1 and LapA2 mRNAs shared more than 93% identity. The deduced amino acid sequences of LapA cDNA clones and in vitro translation of LapA1 mRNA indicated that LAP-A was synthesized as a 60-kDa precursor protein. The processing of a 60-kDa preLAP-A into the mature 55-kDa LAP-A was demonstrated in vivo by expression of the full-length LapA1 cDNA in insect cells. Sequencing of a single LAP-A form isolated from a two-dimensional polyacrylamide gel indicated that LAP-A proteins had two different N termini that were separated by two residues. The LAP-A presequence had features similar to chloroplast transit peptides. Comparison of LAP-A levels in chloroplast and total protein extracts from methyl jasmonate-treated leaves indicated that a small proportion of the LAP-A proteins was detected in the plastids. Inspection of the LAP-A presequence indicated the presence of a dibasic protease (Kex2/furin) processing site motif 6-8 residues upstream from the LAP-A N termini. Its potential role in LAP-A precursor biogenesis is discussed.

Amino Acid Sequence↗
PubMed1977

On the quaternary structure of leucine aminopeptidase.

Small crystals of leucine aminopeptidase were prepared in 2 percent ammonium molybdate. Single molecules were contrasted with 2 percent potassium silico tungstate which gave better contrast and preservation of enzyme activity than ammonium molybdate. The six subunits of the enzyme consist of a spheroidal "head" and some "tail-like" material, which connects the "heads" in pairs. The subunits are arranged at the vertices of either a right triangular prism or of an oblique prism twisted by 42 degree (symmetry 32).

Animals↗
PubMed2002

Leucine aminopeptidase from Streptomyces hygroscopicus is controlled by a low molecular weight inhibitor.

In culture filtrate of Streptomyces hygroscopicus a producer of polyketide antibiotics, a leucine aminopeptidase and its autogenous inhibitor were detected. The leucine aminopeptidase was purified 4573-fold with yield of 82% by combination of ion exchange and hydrophobic chromatography. The enzyme is monomeric with a molecular mass of 51 kDa determined by gel chromatography and 67 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimal activity was at pH 8.0 and 40 degrees C. The pI of leucine aminopeptidase is 8.2. The enzyme is strongly inhibited by 1,10-phenantroline, amastatin and dithiothreitol. Atomic absorption spectrometry indicated 2 mols of ion zinc per mol of enzyme. The enzyme is stable at up to 70 degrees C. Leucine aminopeptidase prefers leucine and methionine as N-terminal amino acids. Activity of leucine aminopeptidase is strongly modulated by an autogenous low-molecular weight inhibitor during fermentation, especially during periods of intensive antibiotic production.

Journal Article↗
PubMed1984

Leucine aminopeptidase from bovine lens and hog kidney. Comparison using immunological techniques, electron microscopy, and X-ray diffraction.

The crystallization of leucine aminopeptidase from hog kidney is reported for the first time. The crystals which diffract to 4-A resolution have the space group P2(1)2(1)2(1) (a = 186.3 A, b = 223.2 A, and c = 80.5 A) and contain four hexamers per unit cell, or one per asymmetric unit. Electron micrographic images of hog kidney leucine aminopeptidase are indistinguishable from micrographs of beef leucine aminopeptidase taken under the same conditions (10). These reveal an equilateral triangle of about 85 A per side, seemingly made of three 40-A diameter spheres. This triangle is circumscribed by another concentric, less-dense triangle of 120 A per side which is rotated 60 degrees with respect to the inner triangle. Immunodiffusion and microcomplement fixation assays indicate that the two enzymes share greater than 90% amino acid sequence homology. This similarity is corroborated by peptide maps of tryptic fragments of the radioiodinated enzymes. The model of the quaternary structure proposed to explain the appearance of electron micrographs of single molecule and crystalline bovine lens enzyme also describes the hog kidney enzyme equally well. That the model of leucine aminopeptidase originally proposed for the beef enzyme also can be used to describe hog kidney leucine aminopeptidase crystal packing in the highly anisometric unit cell provides further corroboration that leucine aminopeptidase in these two species is a hexamer based on two trimers each made of three bilobal promoters.

Animals↗
PubMed1979

A comparison of angiotensinase and placental leucine aminopeptidase during normal pregnancy.

1. Serum leucine aminopeptidase activity measured by its action on hydrolysis of L-leucyl-beta-naphthylamide was increased progressively as pregnancy advanced. Angiotensinase activity determined by bioasaay showed parallel increase during normal pregnancy. 2. The effects of heat treatment and L-methionine on the activity of angiotensinase were compared with those on leucine aminopeptidase activity in pregnancy serum.

Endopeptidases↗
PubMed1987

An improved spectrophotometric assay for leucine aminopeptidase.

A sensitive assay to determine the activity of leucine aminopeptidase (EC 3.4.11.1), using L-leucine thiobenzyl ester as substrate, was developed. Hydrolysis of the ester by leucine aminopeptidase can be monitored in the presence of 5,5-dithiobis-(2-nitrobenzoic acid) by continuous spectrophotometric measurement at 412 nm. Comparison with some amide substrates showed that the thiol ester provides a much more sensitive assay, its specificity constant (Vmax./Km) being some 3000-fold higher than that of leucine p-nitroanilide.

Animals↗
PubMed1984

Possible exposure of leucine aminopeptidase on the cell surface of rabbit blood neutrophils by digitonin treatment.

The sensitivity of leucine aminopeptidase to diazotized sulfanilic acid (DSA) was compared between neutrophils from blood and peritoneal exudates of rabbit. The leucine aminopeptidase activity of peritoneal neutrophils was inhibited about 40% by DSA, whereas that of blood neutrophils was not inhibited at all by the reagent. However, pretreatment of blood neutrophils with digitonin in the presence and in the absence of divalent cations rendered leucine aminopeptidase sensitive to DSA to the same extent as peritoneal neutrophils, without affecting the cell viability and lactate dehydrogenase activity. These findings seem to indicate that the leucine aminopeptidase of blood neutrophils, which is normally inaccessible to DSA, was exposed on the cell surface by digitonin treatment.

Animals↗
PubMed1966

Leucine aminopeptidase in extracts of swine muscle.

1. Leucine aminopeptidase (EC 3.4.1.1) has been demonstrated in swine muscle at a level of activity one-fifth that of the swine kidney. 2. The enzyme has been purified 110-fold by precipitation with ammonium sulphate, heat treatment and chromatography on Sephadex G-100. 3. The enzyme is heat-stable, but is rapidly inactivated below pH7. It requires Mg(2+) or Mn(2+) for activity. The Michaelis constant for leucine amide with Mg(2+)-activated enzyme is 5.0x10(-3)m. 4. Muscle leucine aminopeptidase is very similar to the kidney enzyme.

Animals↗
PubMed1985

A continuous method for the determination of leucine aminopeptidase in human serum with L-leucinamide as substrate.

A continuous procedure for the determination of leucine aminopeptidase is described. L-leucinamide is used as substrate and the liberated ammonia is determined with the glutamate dehydrogenase reaction. The enzyme is Mn2+-activated and 30 mumol/l MnCl2 is necessary for an optimal activity measurement. Influence of buffer type, buffer concentration and pH are reported together with the apparent Km values of leucine aminopeptidase for L-leucinamide and of glutamate dehydrogenase for 2-oxoglutarate. Amastatin, a potent inhibitor, inhibits the reaction of leucine aminopeptidase completely, whereas it has no inhibitory effect on the reaction with glutamate dehydrogenase. The normal reference interval for leucine aminopeptidase is 12-65 U/l at 37 degrees C. The properties of the enzyme are discussed.

Adenosine Diphosphate↗
PubMedDate not supplied

Purification of a leucine aminopeptidase from Eimeria falciformis.

A leucine aminopeptidase was purified from the oocysts of Eimeria falciformis using affinity chromatography and gel filtration techniques. It had a molecular weight of 45-50 kDa. Its maximal activity against leucyl-p-nitro anilide was at pH 8.6. It is a metallo-enzyme highly inhibited by bestatin.

Animals↗
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↗