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PubMed2006

Binding structure of the leucine aminopeptidase inhibitor microginin FR1.

Natural bioactive compounds are of general interest for pharmaceutical research because they may serve as leads in drug development campaigns. Among them, microginins are linear peptides known to inhibit various exopeptidases. The crystal structure of microginin FR1 from Microcystis sp. bound to bovine lens leucine aminopeptidase was established at 1.73 Angstrom resolution. The observed binding structure could be beneficial for the design of potent aminopeptidase inhibitors.

Amino Acid Sequence↗

PubMed2004

Purification and characterization of aminopeptidase (pumAPE) from Ustilago maydis.

The aminopeptidase pumAPE was purified from the haploid fungus Ustilago maydis FB1 strain. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included anion-exchange, hydrophobic interaction, and gel filtration chromatography, resulting in a 23% recovery. The molecular mass of the dimeric enzyme was estimated to be 110 kDa and 58 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 7.0 and at 35 degrees C toward Lys-pNA and the pI was determined to be 5.1. The enzyme was inhibited by EDTA-Na2, 1,10- phenanthroline, bestantin, PMSF and several divalent cations (Cu2+, Hg2+ and Zn2+). The aminopeptidase showed a preference for lysine and arginine in the N-position. The K(m) value was 54.4 microM and the Vmax value was 408 micromolmin(-1)mg(-1) for Lys-pNA.

Aminopeptidases↗

PubMed2004

Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum.

An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling.

Absorption↗

PubMed2005

Crystal structures of the tricorn interacting factor F3 from Thermoplasma acidophilum, a zinc aminopeptidase in three different conformations.

The tricorn interacting factor F3 is an 89 kDa zinc aminopeptidase from the archaeon Thermoplasma acidophilum. Together with the tricorn interacting factors F1 and F2, F3 degrades the tricorn protease products and thus completes the proteasomal degradation pathway by generating free amino acids. Here, we present the crystal structures of F3 in three different conformations at 2.3 A resolution. The zinc aminopeptidase is composed of four domains: an N-terminal saddle-like beta-structure domain; a thermolysin-like catalytic domain; a small barrel-like beta-structure domain; and an alpha-helical C-terminal domain, the latter forming a deep cavity at the active site. Three crystal forms provide snapshots of the molecular dynamics of F3 where the C-terminal domain can adapt to form an open, an intermediate and a nearly closed cavity, respectively. With the conserved Zn(2+)-binding motifs HEXXH and NEXFA as well as the N-terminal substrate-anchoring glutamate residues, F3 together with the leukotriene A4 hydrolase, represents a novel gluzincin subfamily of aminoproteases. We discuss the functional implications of these structures with respect to the underlying catalytic mechanism, substrate recognition and processing, and possible component interactions.

Amino Acid Sequence↗

PubMed2005

Substrate access to the active sites in aminopeptidase T, a representative of a new metallopeptidase clan.

Aminopeptidase T (AmpT) from Thermus thermophilus is a metalloexopeptidase with no similarity to prototypical metallopeptidases with an HExxH or HxxEH motif. The crystal structure of the Staphylococcus aureus homologue of AmpT, which is known as aminopeptidase S (AmpS), has been reported recently. This structure revealed a dimeric protein with a very unusual, elongated shape and a large internal cavity. The active sites were found on the inner walls of the cavity and were entirely shielded from the environment, which suggested either that the dimer in the crystals was not physiologically relevant, or that an inactive conformation had been crystallized. Here, we show by gel-filtration and analytical ultracentrifugation that AmpT, like AmpS, forms dimers in solution, and we present the structure of AmpT in a crystal form with five protomers in the asymmetric unit. The five protomers take conformations that range from fully closed, as in the AmpS structure, to nearly open, so that the active site is almost directly accessible. The different conformations indicate flexibility between the AmpT N and C-domains, and explain how AmpT can be active, although the unusual AmpS dimerization mode applies to AmpT as well.

Aminopeptidases↗

PubMed2005

Distribution of aminopeptidase P like immunoreactivity in the olfactory system and brain of frog, Microhyla ornate.

The enzyme aminopeptidase P (AP-P) is encountered in diverse vertebrate and invertebrate phyla and is known to act on proteins and peptides by releasing their N-terminal amino acid when the penultimate amino acid is proline. The present study is the first attempt at visualizing distribution of this polypeptide in the brain of a vertebrate species. The distribution of this enzyme was studied immunocytochemically in the forebrain of frog Microhyla ornata using antisera directed against cytosolic aminopeptidase P (DAP-P) of Drosophila melanogaster. Receptor cells in the olfactory epithelium exhibited strong AP-P like immunoreaction (ir). Immunoreactive fibers arising from the olfactory epithelium as well as vomeronasal organ joined the olfactory nerve, entered into the olfactory bulb, or accessory olfactory bulb and terminated in distinct glomerular formations. Some immunoreactive fibers traveled caudally and terminated in discrete areas in the telencephalon or diencephalon. Strong AP-P-ir was also seen in the cells of pars intermedia and pars distalis of the pituitary. The pattern of immunoreactivity suggests a role for AP-P in the processing of olfactory information and in hypophysial regulation.

Aminopeptidases↗

PubMed2004

Expression and purification of rat recombinant aminopeptidase B secreted from baculovirus-infected insect cells.

Aminopeptidase B (Ap-B) is a ubiquitous enzyme and its physiological function still remains an open question. This Zn2+ -exopeptidase catalyzes the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. In addition, the enzyme exhibits a residual capacity to hydrolyze leukotriene A4 (LTA4) into the pro-inflammatory lipid mediator leukotriene B4 (LTB4) in vitro. This potential bi-functional nature of Ap-B is supported by a close structural relationship with LTA4 hydrolase, which hydrolyzes LTA4 into LTB4, in vivo, and exhibits an aminopeptidase activity, in vitro. Structural studies are necessary for the detailed understanding of the bi-functional enzymatic mechanism of Ap-B. In this study, we report cDNA cloning, baculovirus expression, and purification of the rat Ap-B (rAp-B). The Ap-B cDNA was constructed from extracted rat testes total RNA and introduced into the pBAC1 baculovirus transfer vector to generate recombinant baculoviruses. rAp-B expression, with or without COOH-hexahistidine tag, was tested in two different insect cell hosts (Sf9 and H5). The enzyme is secreted into the insect cell culture medium, which allowed a rapid purification of the protein. The His-tagged rAp-B was purified using metal affinity resin while the native recombinant rAp-B was partially purified using a single step DEAE Trisacryl ion exchange column. Although the recombinant rAp-B exhibits biochemical properties equivalent to those of the rat testes purified protein, the presence of the histidine-tag seems to partially inhibit the exopeptidase activity. However, this report shows that baculovirus-infected cells are a useful system to produce rat Ap-B for use in studying enzymatic mechanisms in vitro and 3D structure.

Aminopeptidases↗

PubMed2006

Expression, purification, and characterization of an aminopeptidase (Xac2987) with broad specificity from Xanthomonas axonopodis pv. citri.

We report here, the cloning, expression, and purification of a broad specificity aminopeptidase from Xanthomonas campestris pv. citri in fusion with a hexa-histidine tag at the N-terminal portion of the protein to facilitate purification. The protein was expressed in the soluble fraction and could be purified in one step by IMAC, yielding approximately 50mg pure protein per liter of cells. We show that the protein is folded and presents aminopeptidase activity against synthetic substrates. Also, we present the characterization of its specificity, showing that the protein was, indeed, able to catalyze the removal of N-terminal residues from synthetic substrates.

Amino Acid Sequence↗

PubMed2005

Leucine aminopeptidase M inhibitors, cyanostatin A and B, isolated from cyanobacterial water blooms in Scotland.

Two leucine aminopeptidase M inhibitors, cyanostatin A and B, were isolated from cyanobacterial water blooms at Loch Rescobie in Scotland, and specifically from a Microcystis species. Both inhibitors were lipopeptides containing 3-amino-2-hydroxydecanoic acid and weak inhibitors of protein phosphatase (PP2A). Both strongly inhibited the activity of leucine aminopeptidase M with IC50 values of 40 and 12 ng/ml, respectively.

Anabaena↗

PubMed2005

Placental leucine aminopeptidase (P-LAP) and glucose transporter 4 (GLUT4) expression in benign, borderline, and malignant ovarian epithelia.

OBJECTIVE: Increased glucose consumption is a characteristic of malignant cells. Glucose is transported into the cell via facilitative glucose transporters, which are known to be members of a supergene family. The insulin-responsive GLUT4 isoform is expressed almost exclusively in insulin target tissues. P-LAP is a cell surface aminopeptidase, and is a synonym for oxytocinase. P-LAP is also referred to as insulin-regulated membrane aminopeptidase (IRAP) associated with GLUT4-containing vesicle. The authors evaluated P-LAP and GLUT4 expression in benign, borderline, and malignant ovarian epithelia. METHODS: Histologic sections of formalin-fixed, paraffin-embedded specimens from 11 patients with benign serous or mucinous cystadenomas, 14 patients with serous or mucinous borderline tumors, and 80 patients with epithelial-ovarian adenocarcinomas (29 serous, 17 endometrioid, 14 mucinous, and 20 clear cell adenocarcinomas) were stained for P-LAP and GLUT4 using each polyclonal antibody. Expressions of P-LAP and GLUT-4 in ovarian cancer cells were detected by Western blotting. RESULTS: P-LAP immunoreactivity was detected in 2 of 11 benign cystadenomas. None of the 11 benign ovarian tumors showed any immunoreactivity for GLUT4. Seven of 14 borderline tumors demonstrated P-LAP immunoreactivity, while 5 of 14 borderline tumors demonstrated GLUT4 immunoreactivity. P-LAP was expressed in 23 of 29 in serous, 15 of 17 endometrioid, 13 of 14 mucinous, and all clear-cell adenocarcinomas. The tendency toward increased P-LAP expression with advancing grade was observed in serous adenocarcinomas. GLUT4 was expressed in 13 of 29 serous, 13 of 17 endometrioid, 13 of 14 mucinous, and 18 of 20 clear-cell adenocarcinomas. In invasive carcinomas, there was a direct correlation between P-LAP immunoreactivity and GLUT4 immunoreactivity (correlation coefficient [r] = 0.58; P < 0.01). Furthermore, P-LAP overexpression in SKOV3 cells induced the GLUT4 expression. CONCLUSIONS: P-LAP and GLUT4 are available not only for the evaluation of ovarian epithelial malignancy, but also as targets for molecular therapy. Further study to investigate the roles of P-LAP and GLUT4 in ovarian carcinoma is needed.

Adult↗

PubMed2003

Relationship of chloroquine-induced redistribution of a neutral aminopeptidase to hemoglobin accumulation in malaria parasites.

To study the relationship between neutral aminopeptidase activity and hemoglobin accumulation in malaria parasites, we treated mice infected with Plasmodium berghei NYU-2 with chloroquine intraperitoneally in doses ranging from 0.3 to 3 micromol per 25 g mouse. Preparations of infected erythrocytes (normalized to represent 1000 parasites per 1000 erythrocytes) hydrolyzed 1200 nmol of leucine-p-nitroanilide per minute per milliliter of packed erythrocytes, which was 10x more than that of uninfected preparations. The activity in infected preparations was distinguished by resistance to ferriprotoporphyrin IX and puromycin and susceptibility to inhibition by ethanol and Tris. Chloroquine treatment caused the activity in unwashed membrane ghosts of infected preparations to decrease by 50% despite an increase in total activity. Concomitantly, hemoglobin in washed membrane ghosts increased. Electron microscopy revealed that the hemoglobin was retained in endocytic vesicles. Chloroquine-induced redistribution of a neutral aminopeptidase may be the cause of hemoglobin accumulation in endocytic vesicles of malaria parasites.

Aminopeptidases↗

PubMed2003

Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain.

The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.

Aminopeptidases↗

PubMed2003

Rat and mouse membrane aminopeptidase P: structure analysis and tissue distribution.

Membrane-bound aminopeptidase P (mAPP) is a highly specific exopeptidase that removes the N-terminal amino acid only from a peptide (three amino acids or longer) that has a prolyl residue in the second position. mAPP can inactivate bradykinin, a potent vasodilating and cardioprotective peptide hormone, by hydrolyzing the Arg(1)-Pro(2) bond. Studies on the rat have shown that the metabolism of bradykinin is an important physiological role of this enzyme. We report here the complete coding sequences for rat and mouse mAPP determined from mRNA isolated from lung tissue. Key structural features that determine post-translational processing and substrate recognition and catalysis were identified based on sequence homologies and the crystal structure of Escherichia coli aminopeptidase P complexed with Pro-Leu. The tissue-specific expression of mAPP was studied using the polymerase chain reaction. The mAPP gene is widely, but variably, expressed in adult tissues of the rat and mouse and in mouse embryos.

Amino Acid Sequence↗

PubMed2003

High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients.

BACKGROUND: Because non-denaturing electrophoresis and aminopeptidase activity staining often detect noncovalent multi-enzyme complexes, we adopted procedures to specifically detect the aminopeptidase N (APN) molecule itself in liver disease serum. METHODS: Sera or their immunoprecipitate with anti-APN monoclonal antibody were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional electrophoresis and subsequent Western blotting with rabbit anti-APN serum. RESULTS: In all the patient sera examined, the 140-kDa APN isoform was predominant. In all the sera from 10 patients with cholestatic diseases (8 with extra-hepatic cholestasis and 2 with primary biliary cirrhosis), we observed the 260-kDa isoform that was immunoprecipitated with monoclonal APN antibodies and had a similar isoelectric point to the 140-kDa isoform. However, the 260-kDa isoform was observed faintly in 2 out of 12 patients with other liver diseases, including chronic hepatitis and cirrhosis. CONCLUSIONS: We found a novel high-molecular-mass APN isoform (260-kDa) in serum, which is highly likely to be a homodimer of APNs bound covalently and a promising marker of cholestasis. This suggests increased cross-linking reaction between two APN molecules in cholestatic patients.

Aminopeptidases↗

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↗

PubMed1998

Imipramine inhibits soluble enkephalin-degrading aminopeptidase activity in vitro.

Considerable evidence has appeared recently connecting the mechanism of action of some antidepressant drugs with the inhibition of the enzymes responsible for enkephalin degradation. Imipramine in vitro inhibits the enkephalin-degrading aminopeptidase MII and interacts with the enzyme in a mixed competitive-noncompetitive manner. The present work shows that imipramine in vitro also inhibits reversibly soluble enkephalin-degrading aminopeptidase activity in rat brain. Kinetic analysis showed that this enzyme has two different binding sites for the drug, and that imipramine interacts with the enzyme in a mixed noncompetitive-acompetitive way.

Aminopeptidases↗

PubMed1987

Purification and characterization of an aminopeptidase from bovine cornea.

An aminopeptidase from bovine cornea has been extensively purified by gel filtration and ion-exchange chromatography. The purified enzyme with a molecular weight of 96,000 showed broad substrate specificity. All of the various aminoacyl bonds were hydrolyzed optimally at pH 6.5. The purified enzyme showed hydrolytic activity towards bioactive peptides such as enkephalins, bradykinin, and angiotensin-II. The enzyme was inhibited by bestatin, amastatin, puromycin, bacitracin, sulfhydryl reagents and metal chelators. Only Co2+ stimulated the enzyme whereas other heavy metal ions were toxic. The gross properties of the corneal enzyme resemble those of an aminopeptidase III isolated from bovine lens.

Aminopeptidases↗

PubMed1997

Interaction mechanisms of imipramine and desipramine with enkephalin-degrading aminopeptidases in vitro.

In the last few years, considerable evidence has appeared concerning the importance of the opioid systems in the action mechanism of some antidepressant drugs. This action mechanism could be mediated through the inhibition of the enzymes responsible for enkephalin degradation. In this sense, imipramine treatment in vivo increases the enkephalin levels, and this effect is enhanced by inhibitors of enkephalin-degrading enzymes. The present work shows the effects in vitro of imipramine and its active metabolite desipramine on the activities of two membrane-bound enkephalin-degrading aminopeptidases present in rat brain. Imipramine and desipramine in vitro do not affect the aminopeptidase M activity, but they reversibly inhibits the aminoeptidase MII. The enzyme kinetic analysis shows that this enzyme molecule has two different binding sites for each drug, which exert a mixed type enzyme inhibition.

Aminopeptidases↗