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PubMed1985

Ischemia induces partial loss of surface membrane polarity and accumulation of putative calcium ionophores.

To determine if ischemia induces alterations in renal proximal tubule surface membranes, brush border (BBM) and basolateral membranes (BLM) were isolated simultaneously from the same cortical homogenate after 50 min of renal pedicle clamping. Ischemia caused a selective decrease in the specific activity of BBM marker enzymes leucine aminopeptidase and alkaline phosphatase, but did not effect enrichment (15 times). Neither specific activity nor enrichment (10 times) of BLM NaK-ATPase was altered by ischemia. Contamination of BBM by intracellular organelles was also unchanged, but there was an increase in the specific activity (41.1 vs. 60.0, P less than 0.01) and enrichment (2.3 vs. 4.3, P less than 0.01) of NaK-ATPase in the ischemic BBM fraction. Ischemia increased BLM lysophosphatidylcholine (1.3 vs. 2.5%, P less than 0.05) and phosphatidic acid (0.4 vs. 1.3%, P less than 0.05). Ischemia also decreased BBM sphingomyelin (38.5 vs. 29.6%, P less than 0.01) and phosphatidylserine (16.1 vs. 11.4%, P less than 0.01), and increased phosphatidylcholine (17.2 vs. 29.7%, P less than 0.01), phosphatidylinositol (1.8 vs. 4.6%, P less than 0.01), and lysophosphatidylcholine (1.0 vs. 1.8%, P less than 0.05). The large changes in BBM phospholipids did not result from new phospholipid synthesis, since the specific activity (32P dpm/nmol Pi) of prelabeled individual and total phospholipids was unaltered by ischemia. We next evaluated if these changes were due to inability of ischemic cells to maintain surface membrane polarity. Cytochemical evaluation showed that while NaK-ATPase could be detected only in control BLM, specific deposits of reaction product were present in the BBM of ischemic kidneys. Furthermore, using continuous sucrose gradients, the enzymatic profile of ischemic BBM NaK-ATPase shifted away from ischemic BLM NaK-ATPase and toward the BBM enzymatic marker leucine aminopeptidase. Taken together, these data suggest that NaK-ATPase activity determined enzymatically and cytochemically was located within ischemic BBM. We propose that ischemia impairs the ability of cells to maintain surface membrane polarity, and also results in the accumulation of putative calcium ionophores.

Animals↗
PubMed2002

Structure of peptidase T from Salmonella typhimurium.

The structure of peptidase T, or tripeptidase, was determined by multiple wavelength anomalous dispersion (MAD) methodology and refined to 2.4 A resolution. Peptidase T comprises two domains; a catalytic domain with an active site containing two metal ions, and a smaller domain formed through a long insertion into the catalytic domain. The two metal ions, presumably zinc, are separated by 3.3 A, and are coordinated by five carboxylate and histidine ligands. The molecular surface of the active site is negatively charged. Peptidase T has the same basic fold as carboxypeptidase G2. When the structures of the two enzymes are superimposed, a number of homologous residues, not evident from the sequence alone, could be identified. Comparison of the active sites of peptidase T, carboxypeptidase G2, Aeromonas proteolytica aminopeptidase, carboxypeptidase A and leucine aminopeptidase reveals a common structural framework with interesting similarities and differences in the active sites and in the zinc coordination. A putative binding site for the C-terminal end of the tripeptide substrate was found at a peptidase T specific fingerprint sequence motif.

Amino Acid Sequence↗
PubMed2002

Penicillin-resistant Aerococcus viridans bacteremia associated with granulocytopenia.

Aerococcus viridans, a catalase-negative gram-positive coccus rarely causing bacteremia, was isolated from blood cultures of a 52-yr-old man under the granulocytopenic condition. The isolate showed the typical characteristics of A. viridans, i.e., tetrad arrangements in gram stain, positive pyrrolidonyl aminopeptidase (PYR) and negative leucine aminopeptidase (LAP) reactions, and no growth at 45 degrees C. The isolate was revealed to be highly resistant to penicillin, erythromycin, clindamycin, and ceftriaxone, although most strains of A. viridans isolated from the previously reported patients were susceptible to penicillin and other commonly used antibiotics. Even though A. viridans is rarely associated with human infections, it could be a potential causative agent of bacteremia, especially in immunocompromised patients.

Agranulocytosis↗
PubMed2004

Processing of antigenic peptides by aminopeptidases.

Antigenic peptides presented to major histocompatibility complex (MHC) class I molecules are generated in the cytosol during degradation of cellular proteins by the ubiquitin-proteasome proteolytic pathway. Proteasome can generate N-extended precursors as well as final epitopes, and then the precursors are processed to mature epitopes by aminopeptidases. Both cytosolic peptidases (i.e. puromycin-sensitive aminopeptidase, bleomycin hydrolase and interferon-gamma-inducible leucine aminopeptidase) and recently identified metallo-aminopeptidase located in the endoplasmic reticulum (i.e. adipocyte-derived leucine aminopeptidase/endoplasmic reticulum aminopeptidase 1 and leukocyte-derived arginine aminopeptidase) can generate final epitopes from precursor peptides. Some of these aminopeptidases are also considered to destroy certain antigenic peptides to limit the antigen presentation. Taken together, it is getting evident that aminopeptidases located in the cytosol and the lumen of endoplasmic reticulum play important roles in the generation of antigenic peptides presented to MHC class I molecules.

Aminopeptidases↗
PubMed1980

Degradation of melanotropin inhibiting factor by brain.

Degradation of melanotropin inhibiting factor (MIF) was measured by fluorometry, using pareptide as an internal standard, following the separation of the dansyl derivatives of MIF and its metabolites by HPLc. MIF was not split by carboxypeptidases A and B, prolidase, or pyroglutamate aminopeptidase. It was hydrolyzed by leucine aminopeptidase, aminopeptidase M, and carboxypeptidase Y. Rat brain hydrolyzed 159 nmol of MIF per mg of protein per h; the activity was linear with enzyme concentration. Hydrolysis start from the N-terminal end, as shown by the appearance of proline as the first metabolite of the MIF degradation, followed by leucine, glycinamide, leucylglycine, and glycine. Activity in the rat brain regions was in the order striatum, medulla oblongata > cortex, hippocampus, midbrain > hypothalamus, cerebellum, and pituitary. The enzyme was mostly in the supernatant, with significant amounts in the myelin and synaptosomal fractions. MIF aminopeptidase could be separated from carboxypeptidase by centrifugation at 30,000 x g for 20 min and precipitation with 45--75% (NH4)2SO4. It showed pH optima in the alkaline range (8.25 and 8.75) and was inhibited by EDTA, EGTA, SQ 14,225, puromycin, bacitracin, and bestatin.

Aminopeptidases↗
PubMed1991

Growth inhibitory effect of bestatin on choriocarcinoma cell lines in vitro.

Bestatin, one of the biological response modifiers (BRMs), is an inhibitor of aminopeptidase B (AP-B), leucine aminopeptidase (LAP) and aminopeptidase M (AP-M). In this report, we investigated the direct effect of bestatin on the growth of cancer cells in vitro using established four choriocarcinoma cell lines. In vitro chemosensitivity was evaluated by the succinate dehydrogenase inhibition (SDI) test. Bestatin showed the growth-inhibitory effect on all the choriocarcinoma cell lines dose-dependently, especially on NaUCC-4 cells. Both an isomer of bestatin with no inhibitory activity against aminopeptidases, (2R, 3S)-AHPA-(R)-Leu, and another isomer with stronger inhibitory activity against AP-B than bestatin, (2S, 3S)-AHPA-(R)-Leu, did not show growth inhibition on NaUCC-4 cells. So it is suggested that one of the possible mechanisms responsible for the direct action of bestatin on the choriocarcinoma cells may be related to the inhibition of activity of LAP or AP-M rather than that of AP-B. Furthermore, cytotoxicity of actinomycin D on the choriocarcinoma cells was significantly enhanced by combination with bestatin. These results suggest that bestatin has not only an indirect host-mediated anti-tumor activity, but also a direct growth-inhibitory effect on some kinds of cancer cell lines.

Aminopeptidases↗
PubMed1987

Binding, degradation and pressor activity of angiotensins II and III after aminopeptidase inhibition with amastatin and bestatin.

In the metabolism of angiotensin peptides by tissue angiotensinases, aminopeptidases A, B, M and leucine aminopeptidase have been identified as being particularly effective. Because the inhibitory actions of amastatin (AM) and bestatin (BE) are relatively specific for these aminopeptidases, we have examined the effects of these inhibitors on the binding, degradation and pressor activity of angiotensin II (AII) and angiotensin III (AIII). Within 30 min at 37 degrees C, significant metabolism of 125I-AII and 125I-AIII by homogenates of a block of tissue containing hypothalamus, thalamus, septum and anteroventral third ventricle regions of the brain was observed. A majority of 125I-AIII metabolism was due to soluble peptidases, whereas that of 125I-AII primarily resulted from membrane-bound peptidases. AM, BE and reduced incubation temperatures significantly decreased the metabolism of 125I-AII and 125I-AIII. After appropriate adjustments to reflect the proportion of intact radioligand bound, temperature- or inhibitor-induced decreases in metabolism were matched by corresponding increases in specific binding. Heat-treated bovine serum albumin, as a nonspecific peptidase inhibitor, had no effect on either the metabolism or binding of the ligands used. In accordance with their actions in vitro, i.c.v. administration of AM and BE prolonged the pressor activity of subsequently applied AII and AIII. Unexpectedly, the amplitude of the pressor response to AIII was increased by BE, whereas that to AII was decreased by AM. The results of this study indicate that the metabolism of AII and AIII by aminopeptidases is relatively specific and acts to modulate the actions of these peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminopeptidases↗
PubMed1999

Rabbit kidney aminopeptidases: purification and some properties.

Aminopeptidases (EC.3.4.11...) are widely distributed in nature and have medical and biological importance due to their function in the modification and degradation of protein. Two aminopeptidases were purified from rabbit kidney homogenate by ion exchange and gel filtration chromatography columns, using aminoacyl of beta-naphthylamides and p-nitroanilides as substrates. The enzymes' homogeneity was assured by SDS-PAGE. The first enzyme (P1) has an optimum of pH 7.0, a molecular mass of 70 kDa, best catalytical efficiency for methionyl-beta-naphthylamide, is 70% inhibited by 0.5 mM Zn2+ and Co2+ ions, 3.33 mM sodium hydrocortisone succinate and 0.08 mM p-hydroxymercuribenzoate, and is little or not inhibited by EDTA, amino acids, p-nitroaniline, beta-naphthylamine, deoxicholate, bestatin and puromycin. The second enzyme (P2) has an optimum of pH 7.0, a molecular mass of 54 kDa, best catalytical efficiency for Leu-beta-naphthylamide, is inhibited by 0.5 mM ions Zn2+ (45%), 0.02 mM EDTA (94%) 0.08 mM p-hydroxymercuribenzoate (70%), 3.33 mM beta-ME (13%), 1.33 mM p-nitroaniline (40%), 1.33 mM beta-naphthylamine (17%), 1.33 mM sodium deoxicholate (96%), 3.33 mM sodium hydrocortisone succinate (60%), and is 30% activated by 0.5 mM Co2+ ions. Puromycin and bestatin are competitive inhibitors with Ki values in 10(-6) and 10(-7) M order, respectively. P1 is a methionine aminopeptidase, while P2 is a leucine aminopeptidase.

Aminopeptidases↗
PubMedDate not supplied

Differential distribution of placental leucine aminopeptidase/oxytocinase and aminopeptidase A in human trophoblasts of normal placenta and complete hydatidiform mole.

Placental leucine aminopeptidase (P-LAP)/oxytocinase (OTase) degrades several small peptides such as oxytocin (OT), arginine vasopressin (AVP) and angiotensin III (ANGIII), and aminopeptidase A (AP-A) converts angiotensin II (ANGII) to ANGIII. These proteases play an important role in foetal growth and the maintenance of human homeostasis during pregnancy. In this study, we confirmed the distribution of P-LAP and AP-A proteins and messenger RNAs in human trophoblasts in normal placenta and complete hydatidiform mole by immunohistochemical and in-situ hybridization techniques. Immunoreactivity of P-LAP was mainly noted in the apical membrane of syncytiotrophoblasts, and the expression of messenger RNA (mRNA) for P-LAP was predominantly noted in the cytoplasm of syncytiotrophoblastic cells. However, immunoreactivity of AP-A was mainly noted in the apical membrane of cytotrophoblasts and in the basal zone of the syncytiotrophoblasts, and the expression of mRNA for AP-A was predominantly noted in cytoplasm of cytotrophoblastic cells and a little in cytoplasm of syncytiotrophoblastic cells. Thereby, the two proteases were differentially distributed both in normal placenta and hydatidiform mole throughout the gestational age. These results are useful for the further understanding of not only the pathophysiology of pregnancy, but also the pathogenesis of trophoblastic diseases.

Adult↗
PubMed1999

Protective efficacy of trypsin inhibitor on the the gut following rotavirus infection in malnourished infant mice.

Epidemiologic data suggest a close relationship between protein energy malnutrition (or protein energy malnourished) (PEM) and development of infectious diarrheal diseases. In the present studies, the impact of trypsin inhibitor (TI) on the gut following rotavirus (RV) infection was investigated in PEM young growing inbred balb/c mice. The animals were divided into four groups: (1) control; (2) PEM; (3) PEM+RV and (4) PEM+ RV+TI. Control and PEM animals were orally inoculated with normal saline, PEM+RV animals were given 50 microl RV (100ID50) and PEM+RV+TI animals were inoculated with 0.6 mg TI/g body weight + 50 micronl RV. The body weight of each animal was recorded. Six animals from each group were sacrificed on days 0, 1, 3, 5, 7 and 10 post inoculation by cervical dislocation. The intestines were removed, everted and homogenized in ice-cold saline. Leucine aminopeptidase (LAP), glutathione (GSH) levels and L-leucine uptake were determined in the jejunum and ileum. Body weight was decreased in PEM and PEM+RV groups compared to the controls. LAP and leucine uptake levels were elevated in the PEM group but decreased in the PEM+RV group. GSH levels were decreased in the PEM+RV group. Enzyme activity, GSH and LAP uptake levels were restored in the PEM+RV+TI group. The study shows the protective role of soybean TI against RV infection in malnourished mice.

Animals↗
PubMed1994

3-Amino-2-tetralone derivatives: novel potent and selective inhibitors of aminopeptidase-M (EC 3.4.11.2).

Derivatives of 3-amino-2-tetralone were evaluated for their ability to selectively inhibit the membrane-bound zinc-dependent aminopeptidase (EC 3.4.11.2), isolated from porcine kidney. These novel nonpeptidic compounds are potent competitive inhibitors of the enzyme. Some of them have Ki values in the nanomolar range (g, Ki = 80 nM). Moreover, these inhibitors are selective for aminopeptidase-M (AP-M) since they do not inhibit aspartate aminopeptidase and arginine aminopeptidase and only poorly inhibit cytosolic leucine aminopeptidase at high concentrations (g, Ki = 70 microM). The availability of inhibitors which are selective for AP-M with respect to other mammalian aminopeptidases may aid in identifying new endogenous substrates and thus clarify the physiological or pathophysiological role(s) of AP-M.

Aminopeptidases↗
PubMed1996

An intracellular aminopeptidase from Streptomyces rimosus that prefers basic amino acids.

An aminopeptidase from the mycelia of Streptomyces rimosus was isolated in an electrophoretically homogeneous form. It was shown to be a monomeric, acidic protein (pI = 4.4, mol. wt. approx. 83,000), with optimal activity at pH 7.1-7.8 and at 35-41 degrees C. The enzyme was fully inhibited by 0.1 mM EDTA or 1 mM o-phenanthroline; the activity was restored upon addition of 0.05 mM Co2+, Zn2+, or Ni2+. Amastatin, bestatin, and puromycin also inhibited the enzyme. The aminopeptidase hydrolyzed amino-acid-2-naphthylamides and various di- to heptapeptides. The highest catalytic coefficients (23 and 19 microM-1 s-1) were obtained with Arg- and Lys-2-naphthylamide, followed by Leu-, Phe- and Met-derivatives with one order of magnitude lower catalytic coefficients. Basic or bulky hydrophobic amino acids at the P1 and/or P1' position of peptide substrates were preferred. Acidic amino acids and proline were not accepted. The affinity of the enzyme increased with the length of peptide. According to these properties, S. rimosus intracellular aminopeptidase is distinct from the extracellular leucine aminopeptidase of the same organism and can be classified as an Arg(Lys)-preferring metalloaminopeptidase.

Amino Acid Sequence↗
PubMed1996

Identification of a cytoplasm to vacuole targeting determinant in aminopeptidase I.

Aminopeptidase I (API) is a soluble leucine aminopeptidase resident in the yeast vacuole (Frey, J., and K.H. Rohm. 1978. Biochim. Biophys. Acta. 527:31-41). The precursor form of API contains an amino-terminal 45-amino acid propeptide, which is removed by proteinase B (PrB) upon entry into the vacuole. The propeptide of API lacks a consensus signal sequence and it has been demonstrated that vacuolar localization of API is independent of the secretory pathway (Klionsky, D.J., R. Cueva, and D.S. Yaver. 1992. J. Cell Biol. 119:287-299). The predicted secondary structure for the API propeptide is composed of an amphipathic alpha-helix followed by a beta-turn and another alpha-helix, forming a helix-turn-helix structure. With the use of mutational analysis, we determined that the API propeptide is essential for proper transport into the vacuole. Deletion of the entire propeptide from the API molecule resulted in accumulation of a mature-sized protein in the cytosol. A more detailed examination using random mutagenesis and a series of smaller deletions throughout the propeptide revealed that API localization is severely affected by alterations within the predicted first alpha-helix. In vitro studies indicate that mutations in this predicted helix prevent productive binding interactions from taking place. In contrast, vacuolar import is relatively insensitive to alterations in the second predicted helix of the propeptide. Examination of API folding revealed that mutations that affect entry into the vacuole did not affect the structure of API. These data indicate that the API propeptide serves as a vacuolar targeting determinant at a critical step along the cytoplasm to vacuole targeting pathway.

Amino Acid Sequence↗
PubMed2005

Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum.

The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach. T. rubrum genomic and cDNA libraries were screened with Aspergillus spp. and Saccharomyces cerevisiae aminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58-65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 and A. fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities to A. fumigatus orthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T. rubrum in a medium containing keratin as the sole nitrogen source. Synergism between endo- and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.

Amino Acid Sequence↗