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PubMed1992

Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.

Amino Acid Sequence↗

PubMed1985

Partial purification of two distinct enkephalin-degrading aminopeptidases from human cerebrospinal fluid.

In human cerebrospinal fluid, aminopeptidase, dipeptidyl aminopeptidase, dipeptidyl carboxypeptidase, and carboxypeptidase which were capable of hydrolyzing enkephalins were detected. Among these enzymes, two distinct aminopeptidase, designated C-AP1 and C-AP2, were partially purified. These enzymes were not purified thoroughly, but the characteristics of C-AP2 were similar to those of an aminopeptidase purified from monkey brain. But the inhibitory activity of amastatin on C-AP2 was stronger, and that of substance P was negligible. On the other hand, characteristics of C-Ap1 were extremely differ from those of C-AP2 or an aminopeptidase purified from monkey brain. C-AP1 had an optimum pH more in the acidic range (the highest at pH 6.0) and was not inhibited by any of the protease inhibitor tested including bestatin and amastatin.

Aminopeptidases↗

PubMedDate not supplied

Purification and characterization of human seminal plasma aminopeptidase.

A 50.4-fold purification of aminopeptidase is achieved by alcohol precipitation, DEAE-cellulose, CM-cellulose and finally Sephadex G-200 chromatography. On polyacrylamide gel electrophoresis of the purified enzyme after molecular sieving on Sephadex G-200, only one band was obtained, suggesting that the enzyme preparation was obtained almost homogeneous by three steps of column chromatography. Aminopeptidase showed highest activity at pH 7.0, using a buffer system, of 70 mM Na-phosphate. The enzyme was found to be active at 40 degrees C, even at 60 degrees C (80% activity), suggesting that the human seminal plasma enzyme is fairly thermostable. Amongst the various aminoacyl derivatives evaluated as substrates in the present study, L-alanine beta-naphthylamide hydrochloride was found to have the highest rate of hydrolysis. Ovalbumin showed effective cleavage in comparison to that of other natural substrates. The Km value for the purified seminal plasma aminopeptidase towards L-alanine beta-naphthylamide hydrochloride was 4 x 10(-4) M. Hg+2 showed highest inhibitory effect than other metal ions tested in the present study. Concentration causing 50% inhibition of the enzyme (I50) by Hg2+ was 4.7 x 10(-6) M. Inhibition by EDTA at 1 mM concentration in the incubation system was higher than by EGTA and sodium azide, suggesting that the enzyme contains a metallo group at the active site. A 50% inhibition of the enzyme by EDTA was obtained at 5.11 x 10(-3) M. The Ackerman and Potter plot for EDTA inhibition suggests that EDTA is a reversible inhibitor of seminal plasma aminopeptidase. A single molecular form of aminopeptidase was found to be present in human seminal plasma as shown by polyacrylamide activity gel electrophoresis.

Alanine↗

PubMed1982

The effect of two different types of colony-stimulating factor on the expression of aminopeptidase on marrow-derived murine macrophages.

The expression of aminopeptidase, a surface-membrane-bound enzyme, on macrophages formed in liquid cultures of hemopoietic progenitor cells was studied over a period of 20 days. The cultures were stimulated by two biochemically distinct types of colony-stimulating factor (CSF) derived from mouse-lung-conditioned medium (MLCM) and L-cell-conditioned medium (LCCM), respectively. The enzyme content of single cells was determined microphotometrically after staining with Fast Blue B salt and leucine 4-methoxy-2-naphthylamide. In LCCM-stimulated cultures the number of cells expressing aminopeptidase, the enzyme content per cell and the enzyme concentration increased markedly from day 10 onward, while macrophages from MLCM-stimulated cultures only showed borderline yet significantly positive aminopeptidase levels. Maximum enzyme concentrations were found earlier than maximum enzyme content indicating an early local increase in the aminopeptidase concentration on the membrane and subsequently a more uniform distribution over the cell surface. The two types of CSF differ not only in their effect on macrophage production but also in their influence on the expression of the surface enzyme aminopeptidase on these cells.

Aminopeptidases↗

PubMed1993

Possible action of human placental aminopeptidase N in feto-placental unit.

Aminopeptidase N purified from human placenta actively hydrolyzed various immunomodulating peptides from their N-terminus such as splenopentin, thymopentin, thymic humoral factor gamma 2, tuftsin and rigin in vitro. Aminopeptidase N also actively hydrolyzed neuropeptide hormones (met-enkephalin, somatostatin and neurokinin A) and vasoactive peptides (lysyl-bradykinin and angiotensin III) from their N-terminus. In addition, angiotensin II, secretin, thymopoietin II peptide fragment, motilin, endothelin-I and insulin were tested for hydrolysis by aminopeptidase N. Km and Vmax values for the N-terminal amino acid, Thr, a liberation from tuftsin were 267 microM and 8.33 mumol/min/mg protein, respectively. L-Leucyl-p-nitroanilidase activity in the human placental membrane fraction was almost completely neutralized by anti-aminopeptidase N antibody. Our present study suggests that possible roles for surface enzyme aminopeptidase N in the human placenta would be to down-regulate the action of immunomodulating peptides as well as vasoactive and neuropeptide hormones, and to control both immunology and endocrinology of pregnancy.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of an aminopeptidase from Lactobacillus casei subsp. rhamnosus S93.

An aminopeptidase of broad specificity was extracted by cell lysis of a selected strain of Lactobacillus casei subsp. rhamnosus during the late exponential phase. The enzyme was purified 195-fold from crude extract by using an f.p.l.c. system. Native and SDS/PAGE of the purified enzyme showed a single protein band of 89 kDa. The maximum aminopeptidase activity was observed at pH 7.0 and 39 degrees C. The enzyme hydrolysed a range of nitroanilide-substituted amino acids, as well as dipeptides, and accounted for most of the aminopeptidase activity found in cell-free extracts. The enzyme activity was inhibited by metal chelators such as EDTA and 1,10-phenanthroline. Cobalt ions only stimulated aminopeptidase activity and were also able to re-activate the enzyme previously inhibited by metal chelators. The Km and Vmax. values of the aminopeptidase for leucine p-nitroanilide were 0.06 mM and 12.6 mmol/min per mg of protein respectively. This enzyme was stable over the pH range of 5-9 and below 45 degrees C.

Amino Acids↗

PubMed1997

Renal aminopeptidase activities in animal models of hypertension.

Aminopeptidase activity (AP) has been implicated in the metabolism of renal and circulating vasoactive peptides. This activity is involved in the pathogenia of hypertension, essentially in spontaneously hypertensive rats. However, no other animal models, which develop hypertension by other different ways, have been used to study the possible role of aminopeptidase activity. To investigate the role of this activity in the pathogenesis of hypertension, angiotensinase A activity (glutamyl-AP and aspartyl-AP), aminopeptidase M activity (alanyl-AP), aminopeptidase B activity (arginyl-AP), pyroglutamyl-AP, and cystinyl-AP were measured in the serum and kidney of two experimental animal models of renovascular hypertension: Goldblatt two-kidney one clip (G2K-1C) and low renal mass rats (LRM). No differences were found in serum levels of AP in LRM or G2K-1C in comparison with their respective controls. In LRM rats there was a significant decrease in membrane-bound angiotensinase A (glutamyl-AP), arginyl-AP and alanyl-AP activities. In G2K-1C rats there was a significant decrease in soluble and membrane-bound angiotensinase A activity (aspartyl-AP). Our results suggest that AP activities play a role in the regulation of renal vasoactive peptides, and respond differently depending on the cause of hypertension.

Aminopeptidases↗

PubMed1997

Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity.

In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks the conversion of glucose into galactose derivatives. Thus it is possible in the ldl(D) cell line to selectively block O-glycosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose. In this way selectively altered glycosylated forms of the glycoprotein aminopeptidase N can be synthesized and the effects of altered glycosylation can be studied. It is demonstrated that aminopeptidase N carries "mucin-type" O-glycans and that this is predominantly located in the stalk, which connects the catalytic headgroup to the membrane anchor. Normally glycosylated aminopeptidase N is present in the plasma membrane of the ldl(D) cells. This is also the case for the non-O-glycosylated and defectively N-glycosylated forms. This is in line with the finding that the intracellular transport APN is unaffected by the absence of O-glycosylation or by changes in N-glycosylation as the various glycosylated forms of aminopeptidase N are normally converted from the high-mannose form to the complex glycosylated form. Enzymatic activity is not influenced by the changes in glycosylation.

Animals↗

PubMed1998

Aminopeptidase N in sera of healthy subjects is a different N-terminal processed derivative from the one obtained from maternal serum.

A major aminopeptidase present in normal human serum was purified to homogeneity as a 150-kDa molecular species. Western blotting confirmed the binding of an anti-aminopeptidase N antibody to the protein. The N-terminal amino acid sequence of the enzyme was determined. The first 13 amino acids of the enzyme completely matched amino acids 59-71 of the sequence predicted from the human intestinal aminopeptidase N cDNA nucleotide sequence. As reported previously, aminopeptidase N from maternal serum had 68 fewer amino acid residues at the N-terminus than the enzyme obtained from detergent-solubilized membranes. The results indicate that aminopeptidase N in normal serum is a different N-terminal processed derivative from that obtained from maternal serum.

Amino Acid Sequence↗

PubMed1995

Quantification of aminopeptidase N mRNA in T cells by competitive PCR.

The aminopeptidase N (CD13, EC 3.4.11.2) is a well-characterized surface molecule expressed in a variety of cell types and species. Recent data indicate an expression of the APN mRNA and the corresponding aminopeptidase activity in human peripheral T cells and related cell lines as well. Here, the sensitive method of competitive PCR was used to quantify low amounts of APN mRNA in T cell lines. An APN cDNA fragment enshortened by a deletion of 87 bp was used as an internal APN-specific standard. The myelo-monocytic cell line U937 and the lymphoid T cell lines HuT78 and H9 contain 2.3 x 10(7), 5.9 x 10(6) and 5.6 x 10(6) copies/micrograms total RNA, corresponding to 160, 70 and 50 copies/cell, respectively. These data have been confirmed by determination of the APN activity, that represents a fraction only of the total cellular neutral aminopeptidase activity in hematopoetic cells. In the case of the CD13-positive cell line U937, approximately 60-70% of the total neutral aminopeptidase activity could be attributed to APN. In contrast, only a minor fraction (5-20%) of the cellular neutral aminopeptidase activity in the T cell lines H9 and HuT78 represents APN. The results suggest that APN gene expression within the hematopoetic system is not restricted to myelo-monocytic cells, instead a low APN expression may be a common feature of lymphocytes, at least of T cells, too.

Base Sequence↗

PubMed1995

Localization and biosynthesis of aminopeptidase N in pig fetal small intestine.

BACKGROUND & AIMS: Little is known about the expression of brush border enzymes in fetal enterocytes. The aim of this study was to describe the localization and biosynthesis of porcine fetal aminopeptidase N. METHODS: This study was performed using histochemistry and immunoelectron microscopy and [35S]methionine labeling of cultured mucosal explants. RESULTS: Enzyme activity was present in the brush border membrane and extended into the apical cytoplasm. The protein was colocalized with cationized ferritin at the surface of endocytic structures including coated pits, vesicles, tubules, and large vacuoles in the apical cytoplasm. The transient high mannose-glycosylated form of fetal aminopeptidase N was processed to the mature complex-glycosylated form at a markedly slower rate than the enzyme in adult intestine. Likewise, dimerization occurred slowly compared with the adult form of aminopeptidase N, and it took place mainly after the Golgi-associated complex glycosylation. The enzyme had a biphasic appearance in the Mg(2+)-precipitated and microvillar fractions, indicating that the bulk of newly made aminopeptidase N is transported to the brush border membrane before appearing in the apical endocytic structures. CONCLUSIONS: In comparison with the adult enzyme, fetal aminopeptidase N has a more widespread subcellular distribution with substantial amounts present in apical endocytic compartments characteristic of the fetal enterocyte.

Animals↗

PubMed1995

Properties of rat brain dipeptidyl aminopeptidases in the presence of detergents.

Rat brain dipeptidyl aminopeptidases I to IV were assayed in the soluble and membrane-bound fractions of rat brain, and the effects of the detergents Triton X-100 and sodium deoxycholate on their activities were studied. Dipeptidyl aminopeptidases I and II were significantly inhibited in the presence of sodium deoxycholate, but were not affected by the presence of Triton X-100. However, dipeptidyl aminopeptidase III was not influenced by either detergent, whereas the activity of dipeptidyl aminopeptidase IV was stimulated in the presence of Triton X-100, but remained unaffected by deoxycholate. These effects were partially or totally reversed after detergents were removed from the medium with adsorbent polymeric beads. Although detergents may have different effects on each DAP activity, the behavior of each enzyme activity in the presence of these substances was similar regardless of their subcellular location. These findings suggest that, as with other aminopeptidases, each of these proteins corresponds to the same molecular species in two different cell compartments.

Animals↗

PubMed2006

Crystal structure and mechanism of tripeptidyl activity of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis.

The crystal structure of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis was determined. Prolyl tripeptidyl aminopeptidase consists of beta-propeller and catalytic domains, and a large cavity between the domains; this structure is similar to dipeptidyl aminopeptidase IV. A catalytic triad (Ser603, His710, and Asp678) was located in the catalytic domain; this triad was virtually identical to that of the enzymes belonging to the prolyl oligopeptidase family. The structure of an inactive S603A mutant enzyme complexed with a substrate was also determined. The pyrrolidine ring of the proline residue appeared to fit into a hydrophobic pocket composed of Tyr604, Val629, Trp632, Tyr635, Tyr639, Val680, and Val681. There were characteristic differences in the residues of the beta-propeller domain, and these differences were related to the substrate specificity of tripeptidyl activity. The N-terminal amino group was recognized by salt bridges, with two carboxyl groups of Glu205 and Glu206 from a helix in dipeptidyl aminopeptidase IV. In prolyl tripeptidyl aminopeptidase, however, the Glu205 (located in the loop) and Glu636 were found to carry out this function. The loop structure provides sufficient space to accommodate three N-terminal residues (Xaa-Xaa-Pro) of substrates. This is the first report of the structure and substrate recognition mechanism of tripeptidyl peptidase.

Amino Acid Sequence↗

PubMed1999

Binding of Bacillus thuringiensis Cry1Ac toxin to Manduca sexta aminopeptidase-N receptor is not directly related to toxicity.

Bacillus thuringiensis Cry1Ac delta-endotoxin specifically binds a 115-kDa aminopeptidase-N purified from Manduca sexta midgut. Cry1Ac domain III mutations were constructed around a putative sugar-binding pocket and binding to purified aminopeptidase-N and brush border membrane vesicles (BBMV) was compared to toxicity. Q509A, R511A, Y513A, and 509-511 (QNR-AAA) eliminated aminopeptidase-N binding and reduced binding to BBMV. However, toxicity decreased no more than two-fold, indicating activity is not directly correlated with aminopeptidase-N binding. Analysis of toxin binding to aminopeptidase-N in M. sexta is therefore insufficient for predicting toxicity. Mutants retained binding, however, to another BBMV site, suggesting alternative receptors may compensate in vivo.

Acetylgalactosamine↗

PubMed2002

Molecular cloning and analysis of the Cryptosporidium parvum aminopeptidase N gene.

Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules found on the surface of sporozoites and merozoites. In this study, we cloned and expressed the C. parvum aminopeptidase N gene by screening a large insert, P1 artificial chromosome library with a probe identified from a Cryptosporidium genome survey-sequencing project. Analysis of the predicted protein encoded by the 2.3 kb gene demonstrated a high degree of homology with prokaryotic and eukaryotic aminopeptidases. The 783 amino acid sequence predicted a M(r) of approximately 89,000. The active site sequence was found to be highly conserved when compared with other Apicomplexan aminopeptidases. Motifs commonly found in aminopeptidases of this class and a unique single Arg-Gly-Asp (RGD) tripeptide motif predictive of cell adhesion were identified. The aminopeptidase N mRNA was expressed in infective sporozoites and during the infection of human HCT-8 enterocytes as revealed by reverse transcription PCR.

Amino Acid Sequence↗

PubMed2002

Postnatal ontogeny of kinetics of porcine jejunal brush border membrane-bound alkaline phosphatase, aminopeptidase N and sucrase activities.

Our objectives were to determine postnatal changes in the maximal enzyme activity (V(max)) and enzyme affinity (K(m)) of jejunal mucosal membrane-bound alkaline phosphatase, aminopeptidase N and sucrase using a porcine model which may more closely resemble the human intestine. Jejunal brush border membrane was prepared by Mg(2+)-precipitation and differential centrifugation from pigs of suckling (8 days), weaning (28 days), post-weaning (35 days) and adult (70 days) stages. p-Nitrophenyl phosphate (0-8 mM), L-alanine-p-nitroanilide hydrochloride (0-28 mM) and sucrose (0-100 mM) were used in alkaline phosphatase, aminopeptidase N and sucrase kinetic measurements. V(max) of alkaline phosphatase was the lowest in the adult (4.27 micromol.mg(-1) protein.min(-1)), intermediate in the suckling (9.75 micromol.mg(-l) protein.min(-l)) and the highest in the weaning and post-weaning stage (12.83 and 10.40 micromol.mg(-l) protein.min(-l)). K(m) of alkaline phosphatase was high in the suckling and weaning stages (5.14 and 9.93 mM) and low in the adult (0.66 mM). V(max) of aminopeptidase N was low in the suckling (7.04 micromol.mg protein(-1).min(-1)) and high in the post-weaning stage (13.36 micromol.mg(-l) protein.min(-l)). K(m) of aminopeptidase N was the highest in the two weaning stages (2.96 and 3.39 mM), intermediate in the adult (2.33 mM) and the lowest in the suckling stage (1.66 mM). V(max) of sucrase increased from the suckling to the adult (0.48-1.30 micromol.mg(-l) protein.min(-l)). K(m) of sucrase ranged from 11.19 to 16.57 mM. There are dramatic postnatal developmental changes in both the maximal enzyme activity and enzyme affinity of jejunal brush border membrane-bound alkaline phosphatase, aminopeptidase N and sucrase in the pig.

Age Factors↗

PubMed2002

Neutral endopeptidase activity is not elevated in serum in children with cholestatic liver disease: a unique role of aminopeptidase-m in sequential hydrolysis of peptides.

The aim of this study was to determine whether neutral endopeptidase activity is elevated in serum in children with cholestatic liver disease (Alagille syndrome), and whether the enzyme cooperates with the serum aminopeptidase-M in degradation of peptides. Our data suggest that neutral endopeptidase activity remains at a very low level,.undetectable with the assays we have applied, both in the serum from healthy children and those with cholestasis. In contrast, the serum aminopeptidase-M activity is highly increased in cholestasis. We have demonstrated that aminopeptidase-M alone is capable of sequential and complete hydrolysis of enkephalins and low-molecular-weight, nonspecific peptides. The rate of release of free amino acids from both exogenous or endogenous substrate peptides was statistically significantly higher in serum in children with cholestasis compared with healthy children (P < 0.05). Substance P (Ki = 2.5 micromol/liter) and bradykinin (Ki = 27 micromol/liter) were shown to be potent inhibitors of the serum aminopeptidase-M. We postulate that aminopeptidase-M, except when regulating the activity of bioactive peptides, may serve as a scavenger of short, nonspecific peptides in the circulation. Our data seem to provide new insights for further studies on the role of serum peptidases both in physiology and pathophysiology.

Adolescent↗

PubMed1995

Molecular cloning of an insect aminopeptidase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin.

The Bacillus thuringiensis CryIA(c) insecticidal delta-endotoxin binds to a 120-kDa glycoprotein receptor in the larval midgut epithelia of the susceptible insect Manduca sexta. This glycoprotein has recently been purified and identified as aminopeptidase N. We now report the cloning of aminopeptidase N from a M. sexta midgut cDNA library. Two overlapping clones were isolated, and their combined 3095-nucleotide sequence contains an open reading frame encoding a 990-residue pre-pro-protein. The N-terminal amino acid sequence derived from the glycoprotein is present in the open reading frame, immediately following a predicted cleavable signal peptide and a pro-peptide. There are four potential N-linked glycosylation sites. The C-terminal sequence contains a possible glycosylphosphatidylinositol (GPI) anchor signal peptide, which suggests that, unlike most other characterized aminopeptidases, the lepidopteran enzyme is anchored in the membrane by a GPI anchor. This was confirmed by partial release of aminopeptidase N activity from M. sexta midgut brush border membranes by phosphatidylinositol-specific phospholipase C. The deduced amino acid sequence shows significant similarity to the zinc-dependent aminopeptidase gene family, particularly in the region surrounding the consensus zinc-binding motif characteristic of these enzymes.

Amino Acid Sequence↗