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PubMed1977

Post-proline dipeptidyl aminopeptidase (dipeptidyl aminopeptidase IV) from lamb kidney. Purification and some enzymatic properties.

Post-proline dipeptidyl aminopeptidase (dipeptidylpeptide hydrolase, EC 3.4.14.1), also known as glycylprolyl beta-naphthylamidase or dipeptidyl aminopeptidase IV, was isolated and purified in an overall yield of 20% from autolyzed extracts of lamb kidney by CM-cellulose and column chromatography on DEAE-Sephadex and Sephadex G-200. Purified enzyme was homogeneous by disc gel electrophoresis and ultracentrifugal analysis and was most active at pH 7.8 using Gly-Pro beta-napthylamide as substrate. The Km values for Gly-Pro beta-naphthylamide and Ala-Ala beta-naphthylamide were 0.63 and 0.77 mM, respectively. The proline-containing peptides were hydrolysed more than 10-fold faster. By isoelectric focusing a pI of 4.9 was determined. The enzyme was estimated to be 230 000 +/- 15 000 by the sedimentation equilibrium method and sodium dodecyl sulfate polyacrylamide gel electrophoresis indicating that the enzyme is composed of two identical subunits with molecular weights of 115 000. It was inhibited by the active-site directed, irreversible inhibitor diisopropylphosphorofluorofluoridate. Post-proline dipeptidyl aminopeptidase, in contrast to the endopeptidase post-proline cleaving enzyme [9,10] (Walter R. (1976) Biochim. Biophys. Acta 422, 138-158, and Koida, M. and Walter, R. (1976) J. Biol. Chem. 251, 7593-7599) exhibits no endopeptidase activity. Instead it is an exopeptidase with a high specificity for NH2-terminal-free peptides containing a proline residue in the penultimate position and releases the dipeptide with proline being the COOH-terminal moiety. The name "post-proline dipeptidyl aminopeptidase" is suggested.

Animals↗

PubMed1995

Purification and Characterization of a Novel Aminopeptidase, Plastidial Alanine-Aminopeptidase, from the Cotyledons of Etiolated Sugar Beet Seedlings.

During prolonged dark growth of sugar beet (Beta vulgaris L.) seedlings, etioplasts, rapidly after the proplastid-etioplast transition, undergo a degenerative process characterized by ultrastructural modifications, protein loss, and the decrease of carotenoid and chlorophyll accumulation upon illumination. Two plastidial aminopeptidase activities were identified as early markers of this degenerative process (A. El Amrani, I. Couee, J.-P. Carde, J.-P. Gaudillere, P. Raymond [1994] Plant Physiology 106: 1555-1565). The present study focuses on one of these markers and describes the purification to homogeneity and characterization of plastidial alanine-aminopeptidase. This novel aminopeptidase was found to be a metallo-type naphthylamidase particularly active with alanyl, arginyl, and leucyl substrates. Its plastidial location was confirmed by immunofluorescence with polyclonal antibodies against the purified enzyme. Its physico-chemical and enzymic properties are discussed with respect to other higher plant aminopeptidases and to its potential functions during prolonged dark growth.

Journal Article↗

PubMed1981

Aminopeptidases in seeds of picea abies (L.) Karst.: characterization of leucine aminopeptidase by molecular properties and inhibitors.

By either acrylamide or starch gel electrophoresis of Norway spruce (Picea abies) seed extracts, two prominent isoenzyme bands were obtained after staining for leucine aminopeptidase (LAP). These bands were proved to correspond to each other by reelectrophoresis in both gel media. Single endosperm studies with acrylamide gels showed clearly that, in addition to LAP, two bands are expressed after staining for alanine aminopeptidase (AAP) as well. Both the LAP and the AAP activities appeared together as a single peak between catalase and ferritin after gel chromatograhy on Sepharose. Isoelectric focusing in sucrose gradients proved the two LAP activities to have identical isoelectric points revealed that LAP, but not AAP, is detectable by standard starch gel electrophoretic procedures. The two LAP bands refer to approximate molecular weights of 71,000 and 131,000, respectively. Disaggregation studies did not conclusively determine whether these two bands represent different enzymes or not. only inhibitors succeeded in producing a definite differentiation by selective inhibition of one of the two bands. It is concluded that in both gel media the isoenzyme bands reflect the activity of two distinct leucine aminopeptidases.

Aminopeptidases↗

PubMed2001

Study of asparagine 353 in aminopeptidase A: characterization of a novel motif (GXMEN) implicated in exopeptidase specificity of monozinc aminopeptidases.

Aminopeptidase A (EC 3.4.11.7, APA) is a 160 kDa membrane-bound zinc enzyme that contains the HEXXH consensus sequence found in members of the zinc metalloprotease family, the zincins. In addition, the monozinc aminopeptidases are characterized by another conserved motif, GXMEN, the glutamate residue of which has been shown to be implicated in the exopeptidase specificity of aminopeptidase A [Vazeux G. (1998) Biochem. J. 334, 407-413]. In carboxypeptidase A (EC 3.4.17.1, CPA), the exopeptidase specificity is conferred by an arginine residue (Arg-145) and an asparagine residue (Asn-144). Thus, we hypothesized that Asn-353 of the GXMEN motif in APA plays a similar role to Asn-144 in CPA and contributes to the exopeptidase specificity of APA. We investigated the functional role of Asn-353 in APA by substituting this residue with a glutamine (Gln-353), an alanine (Ala-353) or an aspartate (Asp-353) residue by site-directed mutagenesis. Expression of wild-type and mutated APAs revealed that Gln-353 and Ala-353 are similarly routed and glycosylated to the wild-type APA, whereas Asp-353 is trapped intracellularly and partially glycosylated. Kinetic studies, using alpha-L-glutamyl-beta-naphthylamide (GluNA) as a substrate showed that the K(m) values of the mutants Gln-353 and Ala-353 were increased 11- and 8-fold, respectively, whereas the k(cat) values were decreased (2-fold) resulting in a 24- and 14-fold reduction in cleavage efficiency. When alpha-L-aspartyl-beta-naphthylamide or angiotensin II were used as substrates, the mutations had a greater effect on k(cat), leading to a similar decrease in cleavage efficiencies as that observed with GluNA. We then measured the inhibitory potencies of several classes of inhibitors, glutamate thiol, glutamine thiol and two isomers (L- or D-) of glutamate phosphonate to explore the functional role of Asn-353. The data indicate that Asn-353 is critical for the integrity and catalytic activity of APA. This residue is involved in substrate binding via interactions with the free N-terminal part and with the P1 carboxylate side chain of the substrate. In conclusion, Asn-353 of the GXMEN motif, together with Glu-352, contributes to the exopeptidase specificity of APA and plays an equivalent role to Asn-144 in CPA.

Amino Acid Sequence↗

PubMed2004

The C-terminal domain of aminopeptidase A is an intramolecular chaperone required for the correct folding, cell surface expression, and activity of this monozinc aminopeptidase.

Aminopeptidase A (APA, EC 3.4.11.7) is a type II integral membrane glycoprotein responsible for the conversion of angiotensin II to angiotensin III in the brain. Previous site-directed mutagenesis studies and the recent molecular modeling of the APA zinc metallopeptidase domain have shown that all the amino acids involved in catalysis are located between residues 200 and 500. The APA ectodomain is cleaved in the kidney into an N-terminal fragment corresponding to the zinc metallopeptidase domain, and a C-terminal fragment of unknown function. We investigated the function of this C-terminal domain, by expressing truncated APAs in Chinese hamster ovary and AtT-20 cells. Deletion of the C-terminal domain abolished the maturation and enzymatic activity of the N-terminal domain, which was retained in the endoplasmic reticulum as an unfolded protein bound to calnexin. Expression in trans of the C-terminal domain resulted in association of the N- and C-terminal domains soon after biosynthesis, allowing folding rescue, maturation, cell surface expression, and activity of the N-terminal zinc metallopeptidase domain. We also show that the C-terminal domain is not required for the catalytic activity of APA but is essential for its activation. Moreover, we show that the C-terminal domain of aminopeptidase N (EC 3.4.11.2, APN) also promotes maturation and cell surface expression of the N-terminal domain of APN, suggesting a common role of the C-terminal domain in the monozinc aminopeptidase family. Our data provide the first demonstration that the C-terminal domain of an eukaryotic exopeptidase acts as an intramolecular chaperone.

Aminopeptidases↗

PubMed1998

Genetic characterization of pepP, which encodes an aminopeptidase P whose deficiency does not affect Lactococcus lactis growth in milk, unlike deficiency of the X-prolyl dipeptidyl aminopeptidase.

We sequenced the pepP gene of Lactococcus lactis, which encodes an aminopeptidase P (PepP), and demonstrated that the X-prolyl dipeptidyl aminopeptidase PepX plays a more important role than PepP in nitrogen nutrition. PepP shares homology with methionine aminopeptidases and could play a role in the maturation of nascent proteins.

Amino Acid Sequence↗

PubMed1994

Histochemical study of aminopeptidase M, aminopeptidase A, and gamma-glutamyltransferase in the nasal cavity of laboratory rodents andman.

The localization of aminopeptidase M (APM), aminopeptidase A (APA) and gama-glutamyltransferase (GGT) activity was studied at light microscope level in the nasal cavity organs of the laboratory rodents (rat, mouse, guinea pig) and human foetuses. All the enzymes were demonstrated histochemically in chloroform-acetone pretreated cryostat sections with application of azocoupling methods (5, 9). These membrane-bound aminopeptidases may participate in the metabolism of peptides in the nasal cavity. They have specific roles as modulators of growth and differentiation of the epithelial cells. The results revealed differences in enzyme patterns between olfactory and respiratory epithelium. GGT seemed to be present only in respiratory epithelium and in the ducts of Bowman's glands. Activity of APM and APA was found mostly in the fibrocytes which adhered to the basal membrane of the epithelium and glands.

Aminopeptidases↗

PubMed2000

Aminopeptidase yscCo-II: a new cobalt-dependent aminopeptidase from yeast-purification and biochemical characterization.

Saccharomyces cerevisiae aminopeptidase yscCo-II (APCo-II) was purified to apparent homogeneity by gel filtration, affinity chromatography and anion-exchange chromatography. APCo-II is an hexameric cobalt-dependent metallo-enzyme with an estimated native molecular mass of 290 kDa. Enzyme activity is only detected in the presence of cobalt ions at pH 7.0. Substrate specificity studies indicate that aminopeptidase yscCo-II cleaves only basic N-terminal residues. PMSF, Cu(2+), 1,10-phenanthroline and bestatin were found to be very strong inhibitors of aminopeptidase yscCo-II activity. Kinetic studies indicated that the enzyme has a similar K(m) and Ka(Co )(activation constant of cobalt) and, following extraction of cobalt from the enzyme, activity was recovered only after cobalt addition.

Aminopeptidases↗

PubMed1992

Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N.

The nucleotide sequence of the pepN gene from Lactococcus lactis encoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated M(r) of 95,368, which agrees with the apparent M(r) of 95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopeptidase. The amino acid sequence of the aminopeptidase of L. lactis was found to be similar to the corresponding enzymes of human, rat and mouse, with almost 30% of the residues identical. Also, a highly conserved area was identified which has similarity with the active site of thermolysin. A zinc-binding site, as well as the catalytic site for PepN, is predicted to lie within this conserved stretch. Putative promoter regions upstream of PepN were confirmed by primer extension analysis.

Amino Acid Sequence↗

PubMed2003

Neuron-specific aminopeptidase and puromycin-sensitive aminopeptidase in rat brain development.

Neuron-specific aminopeptidase (NAP) and the ubiquitous puromycin-sensitive aminopeptidase (PSA) were compared in the rat hippocampus during early development. Hippocampus contains the highest amount of NAP determined by a fast-protein liquid chromatography-aminopeptidase analyzer using Leu beta-naphthylamide as substrate. Both enzymes were found in the hippocampus in all ages. NAP was lower in immature rat; the 19th embryonic-day fetus contained the least. It increased steeply during the prenatal through the early postnatal period, 9-fold by the first month. The rate of increase diminished subsequently, increasing 20% in the second month and 13% in the third. The age-dependent increase in NAP activity was parallel to its protein expression as determined by Western blot. The specific molecular activity (hydrolytic activity/ NAP antigenicity) in newborn, 15-day-old, and 30-day-old rats were 1.00, 0.88, and 1.00, respectively. The PSA developmental profile without linear increase in activity was distinct from NAP. PSA activity was higher than NAP in decreasing order, 100-4 times, during the same development span. Similarly, different growth profiles for NAP and PSA were also found in the primary culture of developing cerebellar granule cells. Puromycin (1-5 microM) blocked neurite outgrowth and caused apoptosis by nonantibiotic effects. Our data suggest that the synaptosome-enriched NAP plays a role in neuron growth, differentiation, and information programming.

Aging↗

PubMed1997

Experimental evidence for the essential role of the C-terminal residue in the strict aminopeptidase activity of the thiol aminopeptidase PepC, a bacterial bleomycin hydrolase.

PepCs isolated from lactic acid bacteria and bleomycin hydrolases of eukaryotic organisms are strict aminopeptidases which belong to the papain family of thiol peptidases. The structural basis of the enzymic specificity of the lactococcal PepC has been investigated by site-directed mutagenesis. The deletion of the C-terminal residue (Ala-435) abolished the aminopeptidase activity, whereas this deletion led to a new peptidase specificity. The enzymic properties of wild-type and mutant PepCs demonstrate that the terminal alpha-carboxy group plays a key role in the strict aminopeptidase activity.

Alanine↗

PubMed1997

Isolation and sequence analysis of a human cDNA clone (XPNPEPL) homologous to X-prolyl aminopeptidase (aminopeptidase P).

A novel human cDNA (XPNPEPL) encoding a protein of 623 amino acids exhibiting 44% sequence identity and 62% sequence similarity to pig kidney X-prolyl aminopeptidase (aminopeptidase P; EC 3.4.11.9) was obtained by reverse transcription/polymerase chain reaction of phytohemagglutinin-stimulated lymphocyte mRNA. Conserved sequences were found with the prokaryotic X-prolyl aminopeptidase encoding gene (pepP). The human gene translation product exhibits a high sequence homology to the Schizosaccharomyces pombe chromosome I hypothetical protein C22G7.01c and to the S. cerevisiae ORF y11029w. Northern blot analysis indicates an ubiquitous expression of the human XPNPEPL sequence.

Aminopeptidases↗

PubMed1989

xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase.

The heritable stability of ColE1 is dependent on a site-specific recombination system which acts to resolve plasmid multimers into monomers. This plasmid stabilizing recombination system requires the presence in cis of the ColE1 cer region, plus at least two trans-acting factors encoded by the xerA and xerB genes of Escherichia coli. The xerB gene has been cloned and sequenced and found to encode a polypeptide with a calculated mol. wt of 55.3 kd. The predicted amino acid sequence of this protein exhibits striking similarity to that of bovine lens leucine aminopeptidase (53 kd). The biological significance of this similarity is corroborated by genetic and biochemical evidence which suggests that xerB is identical to the E.coli and S.typhimurium pepA genes that encode aminopeptidase A.

Amino Acid Sequence↗

PubMed1977

Serum cystine aminopeptidase and leucine aminopeptidase activity in women with benign and malignant uterine and ovarian tumors.

The serum enzymatic activities of cystine aminopeptidase and leucine aminopeptidase were measured in a group of 113 patients of whom 90 had benign uterine or ovarian tumors, and 23 had cancer of the endometrium or ovary. Thirty healthy nonpregnant women and 260 women at different stages of normal pregnancy served as control groups. The presence of pregnancy-specific enzymes in women with uterine or ovarian tumors showed once again that similar processes occur during pregnancy and malignancy. When ovarian or uterine malignancy is suspected on clinical examination, determination of the activities of these enzymes in serum may be of diagnostic value.

Adolescent↗

PubMed1994

Expression of aminopeptidase N on human choriocarcinoma cells and cell growth suppression by the inhibition of aminopeptidase N activity.

We previously found that an aminopeptidase inhibitor, ubenimex (bestatin), had a growth-suppressive effect on choriocarcinoma cell lines in vitro. To clarify the mechanism of this action, we investigated the expression of aminopeptidase N (AP-N/CD13) on choriocarcinoma cells and other human tumor cells. Two choriocarcinoma cell lines, NaUCC-4 and BeWo, had higher AP-N activity than other cell lines (358.8 and 340.2 nmol/h/10(6) cells, respectively), as did human myeloid leukemia cell line, HL-60 (373.8 nmol/h/10(6) cells). These choriocarcinoma and leukemia cell lines with abundant AP-N activity showed much higher sensitivity to bestatin (IC50 = 0.5, 2.1 and 1.0 micrograms/ml, respectively) than the other cell lines. By immunoblotting and immunocytochemical staining, AP-N was detected as an approximately 165-kDa protein and localized on the cell membrane in choriocarcinoma cells. We also examined the effects of two other aminopeptidase inhibitors and three anti-CD13 monoclonal antibodies (MAbs) (WM15, MCS2 and MY7) on the growth of NaUCC-4 cells. Cell growth was markedly suppressed by the AP-N inhibitor actinonin as well as bestatin, but not by the AP-B inhibitor arphamenine. Of the three MAbs, only WM15, which is able to inhibit AP-N activity, suppressed cell growth in a dose-dependent manner. These results indicate that AP-N inhibitors show a growth-suppressive effect, presumably through inhibition of the enzymatic activity of AP-N on tumor cells, and suggest that AP-N may play important roles in the growth of certain tumors, such as choriocarcinoma and leukemia.

Antibodies, Monoclonal↗

PubMed1998

Isolation and characterization of an alanyl aminopeptidase from rat liver cytosol as a puromycin-sensitive enkephalin-degrading aminopeptidase.

Alanyl aminopeptidase (AAP-S) was purified to homogeneity from rat liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 100,000 on Sephacryl S-200 HR and to be 90,000 on SDS-PAGE in the presence of beta-mercaptoethanol. These findings suggested that the enzyme exists as a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Tyr- and Met-MCAs, and moderately hydrolyzed Arg-, Lys-, Leu-, Phe- and Lys-Ala-MCAs at pHs ranging from 7.5to 8.0. The enzyme also hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order for k(cat)/Km values of AAP-S at the optimal pH (pH 7.5) was Lys->Met->Arg->Ala->Leu->Phe->Tyr->Lys-Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, PCMBS, Zn2+, Cd2+, Co2+, Cu2+ and Hg2+, and puromycin. The amino acid sequence of the first 43 residues of the enzyme was determined as Pro1-Glu-Lys-Arg-Pro5-Phe-Glu-Arg-Leu-Pro10-Thr-Glu-Val-Ser-Pro 15-Ile-Asn-Tyr-Ser-Leu20-(Cys)-Leu-Lys-Pro-Asp25-Leu-Leu- Asp-Phe-Thr30-Phe-Glu-Gly-Lys-Leu35-Glu-Ala-Ala-Ala-Gln40 -Val-Arg-Gln-. This N-terminal amino acid sequence is almost identical with those of puromycin-sensitive enkephalin-degrading aminopeptidases in rat and human brains, and the mouse neuroblastoma cell line Neuro2A. These findings suggest that the AAP-S from rat liver cytosol is a puromycin-sensitive aminopeptidase. Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytosol of the rat liver cells.

Amino Acid Sequence↗

PubMed1997

Expression and localization of aminopeptidase A, aminopeptidase N, and dipeptidyl peptidase IV in benign and malignant human prostate tissue.

BACKGROUND: Cell-surface peptidases are ectoenzymes which regulate the access of bioactive peptides to their receptors on cell membranes. Abnormalities in their expression and function result in altered peptide activity which contribute to neoplastic transformation and/or progression. METHODS: Expression of aminopeptidase A (APA), aminopeptidase N (APN, CD13), and dipeptidyl peptidase IV (DPP IV, CD26) was immunohistochemically examined in 20 benign and 33 malignant prostate tissues (19 primaries and 14 metastases). RESULTS: Benign prostatic stroma exhibited no APA, APN, or DPP IV immunoreactivity. Stromal cells surrounding prostatic carcinoma cells demonstrated increased APA expression in 24/33 (73%) of tumors. Benign prostatic epithelial cells strongly expressed APN and DPP IV but not APA. In contrast, APN was expressed in > 80% of tumor cells in 5/33 (15%) of specimens, heterogeneously expressed (20-80% of cells positive) in 4/33 (12%) of specimens, and minimally expressed or absent in 24/33 (73%) of tumor specimens, with a similar pattern of expression in primary and metastatic tumors. DPP IV was expressed by > 80% of tumor cells in 18/19 (95%) of primary prostate cancer specimens, but in only 7/14 (50%) of metastases. CONCLUSIONS: These data show that cell-surface peptidases are differentially expressed by normal prostatic stromal and epithelial cells, with increased expression of APA in the stroma surrounding prostate cancer cells, absent APN expression in most tumor cells, and a decreased frequency of DPP IV expression in metastatic tumors. Further studies will elucidate the biological effects of the presence or loss of cell-surface peptidases in the benign and malignant prostate.

Aged↗

PubMed1993

cDNA cloning and expression of human glutamyl aminopeptidase (aminopeptidase A).

The murine B-lymphocyte differentiation antigen BP-1/6C3 has been identified as glutamyl aminopeptidase (E-AP), formerly known as aminopeptidase A, the new gene symbol for which is ENPEP. In mice, the enzyme is found on early B-lineage cells and certain stromal cells of the bone marrow and thymus. This ectopeptidase is also expressed by capillary endothelial cells, placenta, and epithelial cells of the intestine and proximal renal tubules. Here we have used a mouse E-AP cDNA to identify the human counterpart in a kidney library. Sequence comparison of the human and mouse cDNAs reveals approximately 80% homology at both nucleotide and predicted amino acid levels. The nucleotide sequence of human E-AP predicts a type II integral membrane protein of 957 amino acids with an 18-amino-acid aminoterminal intracellular domain, and a 22-amino-acid transmembrane domain. The large extracellular carboxyterminal domain contains the zinc-binding motif typical of zinc-dependent metallohydrolases. When the human E-AP cDNA was placed downstream of the SR alpha promoter in an expression vector and transfected into COS-7 cells, the transfected cells exhibited cell surface E-AP activity. A 4.1-kb transcript could be detected in a variety of human tissues, including heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. However, in representative lymphoid leukemias, E-AP transcripts were restricted to pre-B leukemia and were not found in T- and B-cell leukemias. The cDNA cloning and successful expression of human E-AP will allow more precise analysis of its physiological role(s).

Amino Acid Sequence↗