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PubMedDate not supplied

Histoenzymatic activity of leucine aminopeptidase in normal bone marrow cells.

Human, monkey, cat and rat bone marrow cells were studied for the histochemical localization of leucine aminopeptidase activity. The granulocytes have a weak enzymic activity in the immature cells and a very strong one in the differentiated cells. The lymphopoiesis is characterized by a strong reaction in the lymphoblasts and different degrees of the enzyme activity in the lymphocytes. In the erythropoiesis, the undifferentiated cells are strongly positive, while the erythrocytes are negative. The immature and mature cells of the megakariocytic line show a weak leucine aminopeptidase activity.

Age Factors↗
PubMed1990

Molecular cloning and nucleotide sequence of the aminopeptidase T gene of Thermus aquaticus YT-1 and its high-level expression in Escherichia coli.

Aminopeptidase T (AP-T) is a metallo-dependent dimeric enzyme of Thermus aquaticus YT-1, an extremely thermophilic bacterium. We cloned the AP-T gene from T. aquaticus YT-1 into Escherichia coli using a synthetic oligonucleotide as a hybridization probe. The nucleotide sequence of the AP-T gene was found to encode 408 amino acid residues with GTG as a start codon. The molecular weight was calculated to be 44,820. The AP-T was overproduced in E. coli (about 5% of total soluble protein) when the start codon of the gene was changed from GTG to ATG, and the gene was downstream from the tac promoter. The AP-T expressed in E. coli was heat stable and easily purified by heat treatment (80 degrees C, 30 min). The N-terminal amino acid sequence of AP-T showed similarity with that of aminopeptidase II from Bacillus stearothermophilus.

Amino Acid Sequence↗
PubMed1991

Purification and properties of aminopeptidase C from chicken skeletal muscle.

Aminopeptidase C was purified from fresh chicken skeletal muscle by ammonium sulfate fractionation, and by successive chromatography on DEAE-cellulose, Ultrogel AcA 34, DEAE-cellulose again, and an alanine AH-Sepharose 4B affinity column twice. The purified enzyme migrated as a single band by SDS-PAGE. Aminopeptidase C was purified about 300-fold over the crude extract with a yield of 0.6%. The molecular weight of this enzyme was found to be 185,000 by gel filtration in a Sepharose 6B column and 92,000 by SDS-PAGE. The optimum pH for the hydrolysis of L-leucine beta-naphthylamide was 6.0-7.0, the enzyme being stable in the range of pH 6.5-8.0. The activity of this enzyme was strongly inhibited by EDTA and puromycin, and was high against the beta-naphthylamide derivatives of Lys, Leu, Ala and Met. The enzyme was more active towards tri- and tetrapeptides than towards dipeptides.

Amino Acid Sequence↗
PubMed1992

[Isolation of protoplasts of Xanthomonas rubrilineans and their use in the study of localization of aminopeptidases].

The culture of Xanthomonas rubrilineans was able to synthesize a number of intracellular aminopeptidases. To study localization of the enzymes in the cells, a protoplasting procedure was developed providing the yield of 99.7 per cent. The following subcellular fractions were isolated: periplasmic, cytoplasmic and membranous. It was shown that alanine aminopeptidase was a cytoplasmic enzyme and glutamate peptidase was a membrane-bound enzyme.

Aminopeptidases↗
PubMed1992

[Relation between the morphogenesis of Xanthomonas rubrilineans and biosynthesis of aminopeptidase in the process of growth and development of its producer].

The dynamics of growth and development of Xanthomonas rubrilineans, a culture producing intracellular aminopeptidase, was studied. A difference between the growth rate determined by intensity of the total biomass accumulation and the rate of the culture multiplication was found. The difference was due to the presence of two phases in the culture development during the exponential growth: the phase of increasing the linear sizes of the cells and the phase of the culture intensive multiplication. The most intensive synthesis of aminopeptidase was observed during the phase of increasing the linear sizes of the cells. The dynamics of consumption of the main sources of carbon and nitrogen by the culture was investigated.

Aminopeptidases↗
PubMed1992

Chemical modification of the monkey brain cytosolic aminopeptidase that cleaves enkephalin to its constituent amino acids. Evidence for essential tyrosine residue (residues).

A cytosolic monkey brain aminopeptidase is known to cleave amino acids sequentially from the amino termini of peptides like enkephalins, angiotensins, and tuftsin. This enzyme is stimulated by ATP-Mg2+ and inactivated by cyclic AMP-dependent phosphorylation. Chemical modification of this aminopeptidase by several amino acid-modifying agents indicated essential lysine, histidine, arginine, cysteine, and tyrosine residues. The involvement of tyrosine residues in the catalytic activity was confirmed by modification by N-acetylimidazole and acetic anhydride, hydroxylamine treatment, and absorbance studies. Moreover, acetic-1-14C anhydride could label the enzyme that was inhibited by ATP or by bestatin, a competitive inhibitor of the enzyme.

Acetic Anhydrides↗
PubMed1977

Fluorescence of human liver alanine aminopeptidase.

Fluorescence of human liver alanine aminopeptidase has been attributed to tryptophan fluorescence. The fluorescence maximum is at 330 nm, 20 nm lower than that for free tryptophan, suggesting that most of the enzyme tryptophans are in a nonpolar environment and are shielded from solvent. Quenching of enzyme fluorescence by iodide, pyridine, and N-methyl nicotinamide also demonstrates that enzyme tryptophan residues are largely buried and inaccessible to solvent. Those accessible are in negatively charged environments. 8-(1'-dimethylaminonaphthalene-5'-sulfonylamido-octanoic acid (8-DNS-octanoic acid) and epsilon-DNS-L-Lys inhibit aminopeptidase. One molecule of inhibitor when bound to the enzyme quenched 57% and 63% of enzyme fluorescence, respectively. Such efficient quenching may indicate a degree of segregation of tryptophan toward the active center.

Aminopeptidases↗
PubMedDate not supplied

Histochemical study of leucine aminopeptidase in leukemia.

By means of a histochemical method, the leucine aminopeptidase activity was studied in human normal and leukemic peripheral blood. Before administration of cytostatics the enzymic activity was high in the cytoplasm of blasts and in the nuclei of segmented cells. After therapy the activity of leucine aminopeptidase in blasts decreased, while in polymorphs it became negative in nuclei and positive in their cytoplasm. The non-granular leukocytes and platelets displayed a weak enzymic activity both in normal and malignant blood cells. The erythrocytes were negative.

Antineoplastic Agents↗
PubMed2005

Placental leucine aminopeptidase might regulate the effects of oxytocin with resolution in endothelial cells.

BACKGROUND: Oxytocin (OT) is known to cause vascular relaxation via nitric oxide (NO) production and proliferation of endothelial cells. Previously we revealed that human umbilical vascular endothelial cells (HUVECs) expresses placental leucine aminopeptidase (P-LAP)/insulin regulated aminopeptidase (IRAP), which becomes translocation to the cell surface on activation of oxytocin receptor (OTR). However, the physiological roles of P-LAP in HUVECs have not been elucidated. MATERIAL/METHODS: Actions of OT on HUVECs transfected with a copy of the human P-LAP gene were therefore examined with a focus on changes in parameters linked to OTR such as calcium mobilization, NO production and cell proliferation. RESULTS: Cell surface P-LAP activity was significantly elevated (approximately 3 fold) in P-LAP overexpressing-HUVECs and overexpression of P-LAP resulted in remarkable inhibition of OT effects on HUVECs such as cell proliferation, [Ca2+]i, and NO production. CONCLUSIONS: These findings suggested that P-LAP on the plasma membrane in HUVECs regulates the effects of OT with resolution around OTR.

Calcium↗
PubMed2006

[Effect of aminopeptidase inhibitor on differentiation induction activity of all-trans retinoic acid in human acute promyelocytic leukemia NB4 cells and its mechanism].

This study was purposed to investigate whether aminopeptidase inhibitor, bestatin, can potentiate all-trans retinoic acid (ATRA)-inducing differentiation in NB4 cells, and to explore its mechanism. The NB4 cells were exposed to either bestatin and ATRA alone or in combination, the morphological changes of NB4 cells were observed by optical microscopy, the CD11b expression was measured by flow cytometry, the function of defferentiation cells was analyzed by nitroblue-tetrazolium (NBT) reduction assay, the mRNA expressions of c-myc and c-EBPepsilon in NB4 cells were detected by RT-PCR, the c-Myc protein expression was determined by Western blot. The results showed that treatment with bestatin alone induced no significant changes in morphology, NBT reduction activity and CD11b expression in NB4 cells. NB4 cells incubated with 10 nmol/L ATRA plus 100 microg/ml bestatin showed more morphologic feature of metamyelocyte and band neutrophil than ATRA alone treated cells. 100 microg/ml bestatin enhanced the NBT reduction activity in NB4 cells induced by various concentrations of ATRA (10, 20, 40 nmol/L). The effects of various concentrations of ATRA in combination with 100 microg/ml bestatin were statistically different from the effect of ATRA alone (P < 0.01). From 48 to 96 hours, 100 microg/ml bestatin time-dependently increased NBT reduction in NB4 cells induced by 10 nmol/L ATRA (P < 0.01). 10 nmol/L ATRA plus 100 microg/ml bestatin for 72 hours prominently elevated CD11b expression in NB4 cells as compared with ATRA alone treated NB4 cells (P < 0.01). There was a substantial decrease in c-myc mRNA levels when 100 microg/ml bestatin was added to 10 nmol/L ATRA (P < 0.05). Various concentrations (50, 75, 100 microg/ml) of bestatin combined with 10 nmol/L ATRA down-regulated the expression of c-Myc protein, which was negatively correlated with the NBT reduction activity of NB4 cells induced by 10 nmol/L ATRA alone or plus bestatin at various concentrations (r = -0.940, P = 0.017). However, 100 microg/ml bestatin plus 10 nmol/L ATRA could not induce any significant changes in the levels of c-EBPepsilon mRNA as compared with ATRA alone treated NB4 cells. It is concluded that an aminopeptidase inhibitor bestatin can potentiate ATRA-inducing differentiation of NB4 cells, possibly by down-regulating c-myc expression in synergy with ATRA.

Aminopeptidases↗
PubMed1977

[Isolation and properties of leucine aminopeptidase from Aspergillus oryzae].

Homogenious leucine aminopeptidase is purified from "oryzine"--mixture of enzymes produced by surface culture of Asperigillus oryzae using treatment with activated characoal, followed by DEAE-cellulose and hydroxylapatite chromatographies, Biogel P-100 gel-filtration and polyacrylamide-gel electrophoresis. The enzyme has pH optimum 9.0 and the molecular weight 37500 as estimated by gil-filtration through Sephadex G-100 (superfine) and SDS-polyacrylamide gel electrophoresis. Leucine aminopeptidase from Asp. oryzae has a broad substrate specificity, therefore, cleaving with the highest rate the peptides carrying N-terminal leucine. The enzyme is completely inhibited with EDTA and beta-mercaptoethanol, and it is a metalloenzyme.

Aspergillus↗
PubMed1990

Perturbation of intestinal microvillar enzyme biosynthesis by amino acid analogs. Evidence that dimerization is required for the transport of aminopeptidase N out of the endoplasmic reticulum.

The amino acid analogs canavanine, 3-hydroxynorvaline, thialysine, 6-fluorotryptophan, m-fluorotyrosine, and 2-fluorophenylalanine were incorporated into proteins, synthesized in pig intestinal mucosal explants, and their effect on molecular processing and intracellular transport of microvillar enzymes studied. Unless they were used in combination, none of the analogs drastically reduced the expression of aminopeptidase N (EC 3.4.11.2) or sucrase-isomaltase (EC 3.2.1.48, EC 3.2.1.10), but to a varying extent, they all slowed the rate of transport to the apical surface. In contrast, the cellular export of a secretory protein, apolipoprotein A-1, was largely unaffected. For the microvillar enzymes, all six analogs caused an accumulation of the transient, high mannose-glycosylated form, indicating an analog-sensitive stage prior to the Golgi-associated processing. For aminopeptidase N, this arrest was shown to correlate with a reduced ability of its transient high mannose-glycosylated form to form homodimers as judged from cross-linking experiments, suggesting dimerization to be obligatory for transport out of the endoplasmic reticulum.

Amino Acids↗
PubMed1990

A chloride activated alanine aminopeptidase from a melanoma cell line.

Alanine aminopeptidase was partially purified from cultured human melanoma cells (Bowes) by gel filtration on Sephadex G-200 and DEAE Sepharose column chromatography. The molecular weight of the enzyme was about 52,000 as determined by gel filtration on Sephadex G-100. The enzyme hydrolyzed L-alanine beta-naphthylamide (NA), but not or slightly L-methionine-NA, L-leucine-NA, and L-arginine-NA. The Km value for L-alanine-NA was 0.17 mmol/l, pH optimum was 7.4. The enzyme was stable at 50 degrees C for 20 min, but lost about 50% of its activity at 60 degrees C within 20 min. It was markedly stimulated by chloride ions, and was inhibited by sulfhydryl blocking agents and EDTA. The activity was restored by the addition of Co2+ or Zn2+ after EDTA treatment. The enzyme is a metallo- and thiol-dependent and chloride-activated, low-molecular weight aminopeptidase.

Aminopeptidases↗
PubMed1990

Low molecular weight alanine aminopeptidase of human serum: separation and some characteristics.

An alanine aminopeptidase, which has characteristics different from those of known alanine cleaving aminopeptidases, was partially purified from human serum by Sephadex G-200 gel chromatography and DEAE Sepharose column chromatography. The enzyme exhibited a molecular weight of 58,000 by gel chromatography. The pI of the enzyme was 5.0, and it was inactivated at 60 degrees C in 20 min. The enzyme readily hydrolyzed L-alanine beta-naphthylamide, but hardly hydrolyzed the other tested beta-naphthylamides. The Km value for L-alanine beta-naphthylamide was 0.29 mM, the pH optimum 7.5. The activity of the enzyme was enhanced by chloride ions and by sulfhydryl ethylenediaminetetraacetic compounds, and was inhibited by sulfhydryl blocking ethylenediaminetetraacetic agents, and bestatin. Furthermore, 1.10 phenanthroline and ethylenediaminetetraacetic acid were inhibitory, and the activity was restored by CoCl2 and ZnCl2. The enzyme is a chloride-enhanced thiol dependent metalloaminopeptidase.

Aminopeptidases↗
PubMed1990

[Clinical evaluation of urinary glycyl-prolyl dipeptidyl aminopeptidase in patients with urological disease].

Urinary glycyl-prolyl dipeptidyl aminopeptidase (GP-DAP) activity was measured in 18 healthy adults and 252 patients with urological diseases. The GP-DAP activity was significantly higher in patients with prostatic cancer, bladder cancer or renal cancer and also in patients with acute prostatitis or pyelonephritis than in healthy adults. GP-DAP activity was also studied during anticancerous chemotherapy and proved to be a sensitive parameter for renal damage as are urinary N-acetyl-beta-D-glucosaminidase, alanine aminopeptidase, beta 2-microglobulin, alpha 1-microglobulin, and albumin. The analysis of tissue activities suggested that GP-DAP was located not only in the renal parenchyma but also in the prostate and seminal vesicles.

Acetylglucosaminidase↗
PubMed1990

[Intracellular aminopeptidase from Xanthomonas rubrilineans, hydrolyzing alpha-amino acid esters and cefalexin].

The aminopeptidase was isolated from cell-free extracts of Xanthomonas rubrilineans by protein precipitation by isopropyl ester with subsequent purification by affinity chromatography on CABS-Sepharose, bacitracin-Sepharose, gel filtration through Sephadex G-200 and ultrafiltration, the total yield being 32% with 2200-fold purification. The enzyme was homogeneous during SDS-PAAG electrophoresis. Apart from the broad spectrum of the peptidase activity, aminopeptidase possesses a hydrolase activity towards beta-lactam antibiotics and an esterase activity towards L- and D-amino acids. Besides, this enzyme catalyzes the acetyl transfer reaction during cephalexin synthesis from the D-phenylglycine ester and 7-aminodesacetoxycephalosporanic acid. The maximal enzyme activity during L-Ala-pNA and cephalexin hydrolysis is manifested at pH 6.5. The enzyme is stable at pH 4.0-8.0 and is inhibited by o-phenanthroline, p-chloromercuribenzoate, hydrogen acetate and N-bromosuccinimide. The molecular mass of the enzyme is 270-280 kDa. The enzyme is a tetramer; the molecular mass of each of its four subunits is 70 +/- 2 kDa. The isoelectric point for the enzyme is 6.8. The amino acid composition of the enzyme appears as follows: Asp63, Thr33, Ser32, Glu72, Gly55, 1/2Cys3-4, Val45, Ile24, Leu53, Tyr23, Phe24, Lys23, His16, Arg36, Pro60, Met25, Ala55.

Amino Acid Sequence↗
PubMedDate not supplied

[Interhemispherical study of the activities of aminopeptidases, lactate dehydrogenase and glutamate-oxaloacetate transaminase in the rat brain].

Aminopeptidase activity has been assayed from both soluble and membrane bound fractions in eleven regions of the left and right hemispheres of the rat brain, using L-leucine-2-naphthylamide as substrate. Two other metabolic enzymes, lactate dehydrogenase and glutamate-oxalacetate aminotransferase activities were simultaneously determined in the same areas. When individual zones of the left hemisphere were compared with its corresponding right zones, soluble aminopeptidase activities were significantly higher in left frontal cortex and left hypothalamus than in right frontal cortex and right hypothalamus, respectively. No other significant differences were found neither in the remaining areas nor for the other enzymatic activities between both brain hemispheres.

Aminopeptidases↗
PubMed1975

Histochemical demonstration of an LNA-splitting enzyme in the cerebellum of the rat. A aminopeptidase-like reaction localized selectively in the granular layer with acid pH optimum.

Histochemical investigations of leucine aminopeptidase using LNA (L-leucyl-beta-napthylamide) as a substrate reveals a marked enzyme activity selectively localized to the granular layer with inconspicuous reaction in the stratum moleculare and the Purkinje cells in the rat cerebellum. The LNA-splitting enzyme differs from the well-known leucine aminopeptidase (LAP) by its optimum at pH 5.5. The necessary long incubation period used in the present study, and its focal precipitation of the enzyme reaction product in the same place, like acid phosphatases, in the granular layer, suggest a lysosomal localization. The functional role of the LNA-splitting enzyme has been discussed; it is considered that it is involved not only in the protein transformation for synaptic function, but may perhaps also play an important pathogenic role in necrosis, atrophy or even autolysis.

Animals↗