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PubMed1992

Cell surface aminopeptidase A and N activities in human glomerular epithelial cells.

Cell surface aminopeptidases N (APN) and A (APA) have been characterized on cultured human glomerular epithelial cells and a SV40-transformed cell line derived from them. APN had a wide substrate specificity whereas APA only attacked peptides with an acidic N terminal amino acid. Both enzymes also differed by their sensitivity to divalent cations and to aminopeptidase inhibitors. Phorbolmyristate acetate (PMA) stimulated APN but not APA expression after a lag time of 12 hours. An increase of twice the basal value was observed with 10 ng.ml-1 PMA. This effect was confirmed by immunofluorescence staining using a specific anti-APN monoclonal antibody. Both ecto- and total enzyme activities were stimulated by PMA. The effect of PMA was suppressed by H7, a PKC inhibitor, and cycloheximide, an inhibitor of protein synthesis. Thrombin (1 to 2.5 U.ml-1) and interferon (IFN)-gamma (100 U.ml-1) also stimulated APN activity, the latter after longer exposure of the cells. APA activity was increased by 8-bromo-cAMP and two cAMP-stimulating agents, forskolin and isobutylmethylxanthine (IBMX). A twofold increase above basal value was obtained with 100 microM forskolin after 72 hours of treatment. cAMP-stimulated APA activity was suppressed by cycloheximide. Dexamethasone also stimulated APA activity. The effects of forskolin and dexamethasone were additive. These results demonstrate that APN and APA in glomerular epithelial cells are under different regulations: mitogens and IFN-gamma for APN, cAMP and glucocorticoids for APA. This selective expression may imply possible functional consequences in glomerular diseases.

Aminopeptidases↗

PubMed2002

The C-terminal domain, but not the interchain disulphide, is required for the activity and intracellular trafficking of aminopeptidase A.

Mammalian aminopeptidase A (APA; glutamyl aminopeptidase; EC 3.4.11.7) is a type II membrane-spanning protein consisting of a short N-terminal cytosolic domain, a single transmembrane domain and a large extracellular C-terminal domain containing the active site. The extracellular domain consists of a 107 kDa domain, containing the zinc-binding motif and all the residues involved in catalysis, separated by a protease-susceptible hinge region from the 45 kDA C-terminal domain of unknown function. To investigate the role of the 45 kDa domain, a construct of murine APA (G594Delta) lacking this C-terminal domain was expressed in COS-1 cells. This truncated form of APA, although expressed, lacked enzymic activity and failed to reach the cell surface. Confocal immunofluorescence microscopy revealed that G594Delta co-localized with the lectin concanavalin A and had a similar staining pattern as protein disulphide-isomerase, indicating that it was retained in the endoplasmic reticulum. Thus the C-terminal 45 kDa domain appears to be acting like a pro-domain and seems to be required for the correct folding and trafficking of APA. In contrast, mutation of cysteine-43 to serine, which is involved in the disulphide-linkage of the APA homodimer, did not affect the enzymic activity, cellular location or rate of trafficking through the secretory pathway of APA.

Aminopeptidases↗

PubMed1976

Separation of rat muscle aminopeptidases.

By means of chromatography on DEAE-Sephadex, two arylamidases (hydrolysing L-arginine 2-naphthylamide) and three dipeptidyl peptidases (hydrolysing dipeptide 2-naphthylamides) were distinguished in extracts of rat muscle. However, the arylamidase from the larger peak also hydrolysed the dipeptide 2-naphthylamides. Glycyl-L-arginine amide, an alternative substrate for dipeptidyl peptidase I, was not hydrolysed by arylamidase. L-Leucine amide was hydrolysed by an enzyme, presumed to be leucine aminopeptidase, from a separate peak, but was also hydrolysed by arylamidase. Arylamidase, dipeptidyl peptidase III and most of the leucine aminopeptidase could be extracted from the muscle with a neutral salt solution, but dipeptidyl peptidase I was extracted only in the presence of Triton X-100; dipeptidyl peptidase II showed an intermediate extraction behaviour.

Aminopeptidases↗

PubMed1986

Removal of N-terminal methionine from haemoglobin nascent peptides by a membrane-bound rat liver methionine aminopeptidase.

A membrane-bound aminopeptidase able to remove methionine from haemoglobin nascent peptides is described. The enzyme also hydrolyses methionine from methionyl-lysyl-bradykinin but not lysine from lysyl-bradykinin. The tripeptide Met-Ala-Ser is poorly hydrolysed. This aminopeptidase also splits amino acid 2-naphthylamides, being, however, less specific with respect to these synthetic substrates.

Amides↗

PubMed1998

Increased intracellular sequestration of the insulin-regulated aminopeptidase upon differentiation of 3T3-L1 cells.

In fat and muscle cells, the glucose transporter GLUT4 is sequestered in an intracellular compartment under basal conditions and redistributes markedly to the plasma membrane in response to insulin. Recently, we characterized a membrane aminopeptidase, designated IRAP (insulin-regulated aminopeptidase), that colocalizes with intracellular GLUT4 and similarly redistributes markedly to the plasma membrane in response to insulin in adipocytes. In contrast to GLUT4, IRAP is also expressed in 3T3-L1 fibroblasts, and this finding provided an opportunity to compare its subcellular distribution in fibroblasts and adipocytes. The relative amount of IRAP at the cell surface was measured by a cell surface biotinylation method. The portion of total IRAP at the cell surface in unstimulated adipocytes was 30% of that in unstimulated fibroblasts. Upon insulin treatment the portion of IRAP at the cell surface was the same in fibroblasts and adipocytes, and was increased 1.8-fold in fibroblasts and 8-fold in adipocytes. A similar analysis of the distribution of the transferrin receptor (TfR), the paradigm for recycling plasma membrane receptors, revealed that the portions of the TfR at the cell surface in both the basal and insulin-treated states were almost unchanged upon differentiation, and that insulin caused an increase of about 1. 6-fold in the amount of TfR at the cell surface. These results show that enhanced intracellular sequestration of IRAP occurs during adipogenesis, and that this effect underlies the larger insulin-elicited fold increase of IRAP at the cell surface in adipocytes.

3T3 Cells↗

PubMed1985

In vitro degradation of oxytocin by pregnancy serum, placental subcellular fractions and purified placental aminopeptidases.

The degradation of oxytocin (formula; see text) by human placental particulate and soluble fractions, pregnant and non-pregnant sera, and purified enzymes such as microsomal placental leucine aminopeptidase (P-LAP), retroplacental serum P-LAP and placental post-proline endopeptidase, was studied by measuring liberated amino acids with a high performance liquid chromatograph (HPLC). While the placental particulate fraction degraded oxytocin almost completely into single amino acid and amide, the placental soluble fraction did not liberate any amino acid and amide. Purified retroplacental P-LAP liberated Tyr2, Ile3, Gln4 and Asn5 from the cyclic structure of oxytocin actively. Pregnancy serum containing the retroplacental P-LAP liberated also these amino acids and amides. Purified microsomal P-LAP liberated Leu8 in addition to these amino acids and amides. Although purified placental post-proline endopeptidase or porcine kidney leucine aminopeptidase (LAP) could not liberate any amino acid and amide from oxytocin, the combination of the post-proline endopeptidase with porcine kidney LAP or with placental microsomal P-LAP actively liberated all amino acids and amides detectable by HPLC. When the ratio of amino acid liberation velocity to LAP activity measured with leu-p-nitroanilide was calculated, the ratios for cyclic amino acids such as Tyr2 and Ile3 were high with the placental particulate fraction, the mixture of post-proline endopeptidase and microsomal P-LAP, retroplacental P-LAP, and pregnancy serum. The ratio for Leu8 was high with the placental particulate fraction and the mixture of post-proline endopeptidase and microsomal P-LAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1993

Aspartate-aminopeptidase activity during the estrous cycle and the pregnancy in rat brain and pituitary gland.

The present report describes the activity of aspartate aminopeptidase, in 9 brain areas and in the pituitary gland, during the estrous cycle and several stages of pregnancy of the rat. The quantitation of the enzyme activity was performed by measuring the rate of hydrolysis of the chromogenic substrate Asp-beta-naphthylamide. Aspartate aminopeptidase activity, in the pituitary gland and in several limbic areas, was found to be greater in cyclic than in pregnant rats. However the enzyme activity did not change significantly during either the estrous cycle and pregnancy.

Aminopeptidases↗

PubMed2006

Inhibitors of Plasmodium falciparum methionine aminopeptidase 1b possess antimalarial activity.

With >1 million deaths annually, mostly among children in sub-Saharan Africa, malaria poses one of the most critical challenges in medicine today. Although introduction of the artemisinin class of antimalarial drugs has offered a temporary solution to the problem of drug resistance, new antimalarial drugs are needed to ensure effective control of the disease in the future. Herein, we have investigated members of the methionine aminopeptidase family as potential antimalarial targets. The Plasmodium falciparum methionine aminopeptidase 1b (PfMetAP1b), one of four MetAP proteins encoded in the P. falciparum genome, was cloned, overexpressed, purified, and used to screen a 175,000-compound library for inhibitors. A family of structurally related inhibitors containing a 2-(2-pyridinyl)-pyrimidine core was identified. Structure/activity studies led to the identification of a potent PfMetAP1b inhibitor, XC11, with an IC(50) of 112 nM. XC11 was highly selective for PfMetAP1b and did not exhibit significant cytotoxicity against primary human fibroblasts. Most importantly, XC11 inhibited the proliferation of P. falciparum strains 3D7 [chloroquine (CQ)-sensitive] and Dd2 (multidrug-resistant) in vitro and is active in mouse malaria models for both CQ-sensitive and CQ-resistant strains. These results suggest that PfMetAP1b is a promising target and XC11 is an important lead compound for the development of novel antimalarial drugs.

Amino Acid Sequence↗

PubMed1995

Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Using partial amino acid sequence data derived from porcine methionyl aminopeptidase (MetAP; methionine aminopeptidase, peptidase M; EC 3.4.11.18), a full-length clone of the homologous human enzyme has been obtained. The cDNA sequence contains 2569 nt with a single open reading frame corresponding to a protein of 478 amino acids. The C-terminal portion representing the catalytic domain shows limited identity with MetAP sequences from various prokaryotes and yeast, while the N terminus is rich in charged amino acids, including extended strings of basic and acidic residues. These highly polar stretches likely result in the spuriously high observed molecular mass (67 kDa). This cDNA sequence is highly similar to a rat protein, termed p67, which was identified as an inhibitor of phosphorylation of initiation factor eIF2 alpha and was previously predicted to be a metallopeptidase based on limited sequence homology. Model building established that human MetAP (p67) could be readily accommodated into the Escherichia coli MetAP structure and that the Co2+ ligands were fully preserved. However, human MetAP was found to be much more similar to a yeast open reading frame that differed markedly from the previously reported yeast MetAP. A similar partial sequence from Methanothermus fervidus suggests that this p67-like sequence is also found in prokaryotes. These findings suggest that there are two cobalt-dependent MetAP families, presently composed of the prokaryote and yeast sequences (and represented by the E. coli structure) (type I), on the one hand, and by human MetAP, the yeast open reading frame, and the partial prokaryotic sequence (type II), on the other.

Amino Acid Sequence↗

PubMed1996

Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release.

Angiotensin (Ang) II and Ang III are two peptide effectors of the brain renin-angiotensin system that participate in the control of blood pressure and increase water consumption and vasopressin release. In an attempt to delineate the respective roles of these peptides in the regulation of vasopressin secretion, their metabolic pathways and their effects on vasopressin release were identified in vivo. For this purpose, we used recently developed selective inhibitors of aminopeptidase A (APA) and aminopeptidase N (APN), two enzymes that are believed to be responsible for the N-terminal cleavage of Ang II and Ang III, respectively. Mice received [3H]Ang II intracerebroventricularly (i.c.v.) in the presence or absence of the APN inhibitor, EC33 (3-amino-4-thio-butyl sulfonate) of the APN inhibitor, EC27 (2-amino-pentan-1,5-dithiol). [3H]Ang II and [3H]Ang III levels were evaluated from hypothalamus homogenates by HPLC. EC33 increased the half-life of [3H]Ang II 2.6-fold and completely blocked the formation of [3H]Ang III, whereas EC27 increased the half-life of [3H]Ang III 2.3-fold. In addition, the effects of EC33 and EC27 on Ang-induced vasopressin release were studied in mice. Ang II was injected i.c.v. in the presence or absence of EC33, and plasma vasopressin levels were estimated by RIA. While vasopressin levels were increased 2-fold by Ang II (5 ng), EC33 inhibited Ang II-induced vasopressin release in a dose-dependent manner. In contrast, EC27 injected alone increased in a dose-dependent manner vasopressin levels. The EC27-induced vasopressin release was completely blocked by the coadministration of the Ang receptor antagonist (Sar1-Ala8) Ang II. These results demonstrate for the first time that (i) APA and APN are involved in vivo in the metabolism of brain Ang II and Ang III, respectively, and that (ii) the action of Ang II on vasopressin release depends upon the prior conversion of Ang II to Ang III. This shows that Ang III behaves as one of the main effector peptides of the brain renin-angiotensin system in the control of vasopressin release.

Amino Acid Sequence↗

PubMed1997

The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2.

The inhibition of new blood vessel formation (angiogenesis) is an effective means of limiting both the size and metastasis of solid tumors. The leading anti-angiogenic compound, TNP-470, has proven to be effective in in vitro and in animal model studies, and is currently being tested in phase III antitumor clinical trials. Despite many detailed pharmacological studies, little is known of the molecular mode of action of TNP-470. Using a derivative of the TNP-470 parent compound, the fungal metabolite, fumagillin, we have purified a mammalian protein that is selectively and covalently bound by this natural product. This fumagillin binding protein was found to be a metalloprotease, methionine aminopeptidase (MetAP-2), that is highly conserved between human and Saccharomyces cerevisiae. In the absence of MetAP-1, a distantly related methionine aminopeptidase, MetAP-2 function is essential for vegetative growth in yeast. We demonstrate that fumagillin selectively inhibits the S. cerevisiae MetAP-2 protein in vivo. The binding is highly specific as judged by the failure of fumagillin to inhibit MetAP-1 in vivo. Hence, these results identify MetAP-2 as an important target of study in the analysis of the potent biological activities of fumagillin.

Amino Acid Sequence↗

PubMed1999

Aminopeptidase A inhibitors as potential central antihypertensive agents.

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental models, such as spontaneously hypertensive rats and transgenic mice expressing both human renin and human angiotensinogen transgenes. We recently reported that, in the murine brain, angiotensin II (AngII) is converted to angiotensin III (AngIII) by aminopeptidase A (APA), whereas AngIII is inactivated by aminopeptidase N (APN). If injected into cerebral ventricles (ICV), AngII and AngIII cause similar pressor responses. Because AngII is metabolized in vivo into AngIII, the exact nature of the active peptide is not precisely determined. Here we report that, in rats, ICV injection of the selective APA inhibitor EC33 [(S)-3-amino-4-mercaptobutyl sulfonic acid] blocked the pressor response of exogenous AngII, suggesting that the conversion of AngII to AngIII is required to increase blood pressure (BP). Furthermore, ICV injection, but not i.v. injection, of EC33 alone caused a dose-dependent decrease in BP by blocking the formation of brain but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol), administered alone via the ICV route, increases BP. This pressor response was blocked by prior treatment with the angiotensin type 1 (AT(1)) receptor antagonist, losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in BP increase, through interaction with AT(1) receptors. These data demonstrate that AngIII is a major effector peptide of the brain RAS, exerting tonic stimulatory control over BP. Thus, APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors as central antihypertensive agents.

Aminopeptidases↗

PubMed2003

Protonation states of methionine aminopeptidase and their relevance for inhibitor binding and catalytic activity.

We have performed a computational study of different protomeric states of the methionine aminopeptidase active site using a combined quantum-mechanical/molecular mechanical simulation approach. The aim of this study was to clarify the native protonation state of the enzyme, which is needed for the development of novel irreversible inhibitors that can possibly be used as antiangiogenic and antibiotic drugs by virtual screening and other drug design methods. The results of the simulations indicated that two protonation states are possible without disturbing the overall geometry of the active site. We then verified experimentally the presence of the two protonation states by studying the substrate hydrolysis and inhibitor binding reactions at different pH values and come to the conclusion that one of the protomeric states is relevant for inhibitor binding, whereas the other is relevant for substrate hydrolysis. This result has implications for the development of other inhibitors of this class of enzymes and adds a new perspective to the pharmacological properties of the antiangiogenic drug fumagillin, which is an irreversible inhibitor of the human methionine aminopeptidase type II.

Aminopeptidases↗

PubMed2003

A single amino acid residue defines the difference in ovalicin sensitivity between type I and II methionine aminopeptidases.

TNP-470, the first anti-angiogenic small molecule to enter clinical trials, targets methionine aminopeptidase-2 (MetAP-2), a metalloprotease that cleaves the N-terminal methionine of proteins. Previously, biochemical binding, in vivo yeast studies, and structural studies of human methionine aminopeptidase-2 bound to TNP-470 and its analogs fumagillin and ovalicin revealed that these compounds exhibit specificity for MetAP-2 over its family member MetAP-1. To further elucidate the nature of this specificity, we developed a yeast-based screen for human MetAP-2 mutations that confer ovalicin resistance. Of the three resistant alleles, A362T appeared in the majority of clones and was found to be the most resistant to the ovalicin class of inhibitors. Alignment of human MetAP-2 with human MetAP-1, which is naturally ovalicin-resistant, revealed that the analogous residue in MetAP-1 is also a threonine. Mutation of this residue to alanine resulted in an ovalicin-sensitive MetAP-1 allele, demonstrating that an alanine at this position is critical for inhibition by ovalicin. These results provide a molecular explanation for the specificity exhibited by this class of anti-angiogenic agents for MetAP-2 over MetAP-1 and may prove useful in the development of additional MetAP-2-specific therapeutic agents.

Alanine↗

PubMed1960

Improvement in the histochemical localization of leucine aminopeptidase with a new substrate, L-leucyl-4-methoxy-2-naphthylamide.

A new method for the histochemical demonstration of leucine aminopeptidase in fresh frozen sections was developed with the substrate L-leucyl-4-methoxy-2-naphthylamide. The superior enzyme localization is due to the more rapid rate of coupling of the hydrolysis product, 4-methoxy-2-naphthylamine as compared to 2-naphthylamine itself, and to the low lipid solubility and high substantivity for protein of the copper chelate of the dye formed on coupling with tetrazotized diorthoanisidine. A comparison of the old and the new method is illustrated, and a description is given of the localization of leucine aminopeptidase in the tissues of the rat and man.

2-Naphthylamine↗

PubMed1985

Variable distribution of aminopeptidase A in male reproductive organs of mammals.

Aminopeptidase A (AP-A) was analysed in the reproductive organs of the boar, bull, gerbil and man. High hydrolysis of alpha-L-glutamyl-beta-naphthylamide (GluNA) and alpha-L-aspartyl-beta-naphthylamide (AspNA) with activation by alkaline earth metals was detected in the ampulla, seminal vesicles, and seminal vesicle secretions of the bull and in the cauda epididymis of the boar and gerbil. In man, weak AP-A activity was found in all reproductive tissues. Histochemically, AP-A was localized in the epithelial cells of tissues having a high specific activity for the enzyme. AP-A was absent from human seminal fluid, whilst bovine seminal fluid had strong, and boar seminal fluid weaker, AP-A activity. Gel filtration of bull seminal vesicle secretions and seminal fluid, boar seminal fluid or an homogenate of boar and gerbil epididymal cauda and human epididymis and seminal vesicles on Sephacryl S-300 resulted in a major high-molecular-weight activity peak A at Ve/Vo = 1.17 and another low-molecular-weight peak B at Ve/Vo = 1.51 (man), 1.62 (boar, bull) or 1.75 (gerbil). This fractionation was not in all cases able to separate AP-A from aminopeptidase(s), which were active on L-alanine-beta-naphthylamide (AlaNA) but showed no activation by alkaline earth metals. Homogenates of bovine epididymis showed only the low-molecular-weight GluNA peak B, but two areas of activity for AlaNA hydrolysis. In bovine seminal vesicles and porcine epididymis, AP-A activity appeared to be linked with the functional maturity of these organs. The high-molecular-weight AP-A (peak A) appeared to be the predominant form in seminal fluid.

Aminopeptidases↗

PubMed1993

Purification and characterization of an aminopeptidase A from Staphylococcus chromogenes and its use for the synthesis of amino-acid derivatives and dipeptides.

An aminopeptidase with original specificity was purified 3800-fold to homogeneity from a cellular extract of Staphylococcus chromogenes. The enzyme was specific for acidic amino acids (Asp and Glu) at the N-terminus of peptides and thus can be classified as an aminopeptidase A. However, its specificity was not restricted to acidic amino acids: alpha-hydroxy acids such as L-malic and L-lactic acids were also accepted in position P1. The enzyme had a broad specificity for the residue at position P' 1, accepting all types of amino acids, including Pro, in this position. The optimal conditions for the hydrolysis of Asp-Phe-NH2 were pH 9.5 and 60 degrees C. The enzyme was inhibited by chelating agents and serine-protease inhibitors. The activity lost by treatment with chelating agents could be restored by Mn2+ or Zn2+ which also stimulated the native enzyme. This suggests that it is a metalloprotease with a serine residue essential for the activity. The native enzyme had an apparent molecular mass of 430 kDa on gradient-gel electrophoresis and subunits of 43 kDa as determined by SDS/PAGE. The enzyme catalyzed the synthesis of peptide and amino acid derivatives such as Asp-Phe-OMe (Aspartame) and malyl-Tyr-OEt from L-Asp and L-malic acid as acyl donors and L-Phe-OMe and L-Tyr-OEt as nucleophiles, respectively. The use of the enzyme as a reagent in protease-catalyzed peptide synthesis, N-terminal protection and subsequent deprotection, is described.

Amino Acid Sequence↗

PubMed1989

Methionine aminopeptidase gene of Escherichia coli is essential for cell growth.

We localized the methionine aminopeptidase (map) gene on the Escherichia coli chromosome next to the rpsB gene at min 4. Genetically modified strains with the chromosomal map gene under lac promoter control grew only in the presence of the lac operon inducer isopropyl-beta-thiogalactoside. Thus, methionine aminopeptidase is essential for cell growth.

Aminopeptidases↗