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PubMed1981

Reconstitution of purified amphiphilic pig intestinal microvillus aminopeptidase. Mode of membrane insertion and morphology.

Pig intestinal microvillus aminopeptidase (EC 3.4.11.2) was reincorporated into lipid membranes by using either beta-octyl glucoside or sodium deoxycholate. The results showed that for this enzyme the deoxycholate-dialysis method was the preferable one. By using this method, microvillus aminopeptidase was inserted almost quantitatively into either phosphatidylcholine vesicles or microvillus-lipid vesicles. By proteolytic treatment of the vesicles, by probing the aminopeptidase with an inhibitory antibody and by monitoring the positioning of the anchor with the aid of [125I]iodonaphthyl azide, it was demonstrated that the catalytically active part was located outside the liposomes and the anchoring peptide(2) was associated with the membrane. Electron-microscopic observation on this model system demonstrated a dimeric symmetrical structure of aminopeptidase (dimensions about 13.5 nm X 5.5 nm) separated by a 5 nm gap from the membrane. This distance corresponds to a molecular weight of 2000-5000 for this junctional segment of the anchor connecting the intramembrane part of the anchor with the catalytically active main part of the aminopeptidase.

Aminopeptidases↗

PubMed1982

Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on aminopeptidase N and sucrase-isomaltase.

The biogenesis of two microvillar enzymes, aminopeptidase N (EC 3.4.11.2) and sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. Both enzymes became inserted into the membrane during or immediately after polypeptide synthesis, indicating that translation takes place on ribosomes attached to the rough endoplasmic reticulum. The earliest detectable forms of aminopeptidase and sucrase-isomaltase were polypeptides of Mr 140 000 and 240 000 respectively. These polypeptides were susceptible to treatment with endo-beta-N-acetylglucosaminidiase H (EC 3.2.1.96), suggesting that the microvillar enzymes during or immediately after completion of protein synthesis become glycosylated with a 'high-mannose' oligosaccharide structure similarly to other plasma-membrane and secretory proteins. After 20--40 min or 60--90 min of chase, respectively, aminopeptidase N and sucrase-isomaltase were reglycosylated to give the polypeptides of Mr 166 000 (aminopeptidase N) and 265 000 (sucrase-isomaltase). These were expressed at the microvillar membrane after 60--90 min. During the entire process of synthesis and transport to the microvillar membrane the enzymes were bound to membranes, indicating that the biogenesis of aminopeptidase N and sucrase-isomaltase occurs in accordance with the membrane flow hypothesis.

Acetylglucosaminidase↗

PubMed1990

Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme.

Aminopeptidase P (EC 3.4.11.9) was solubilized from pig kidney membranes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and then purified by a combination of anion-exchange and hydrophobic-interaction chromatographies. Contaminating peptidase activities were removed by selective affinity chromatography. The purified enzyme was apparently homogeneous on SDS/PAGE with an Mr of 91,000. Enzymic deglycosylation revealed that aminopeptidase P is a glycoprotein, with up to 25% by weight of the protein being due to the presence of N-linked sugars. The phospholipase-solubilized aminopeptidase P was recognized by an antiserum to the cross-reacting determinant (CRD) characteristic of the glycosyl-phosphatidylinositol anchor. This recognition was abolished by mild acid treatment or deamination with HNO2, indicating that the CRD was due exclusively to the inositol 1,2-cyclic phosphate ring epitope generated by the action of PI-PLC. The activity of aminopeptidase P was inhibited by chelating agents and was stimulated by Mn2+ or Co2+ ions, confirming the metallo-enzyme nature of this peptidase. Selective inhibitors of other aminopeptidases (actinonin, amastatin, bestatin and puromycin) had little or no inhibitory effect.

Aminopeptidases↗

PubMed1993

A role for aminopeptidase N in Na(+)-dependent amino acid transport in bovine renal brush-border membranes.

A monoclonal antibody FD19 which removes reconstitutable Na(+)-dependent amino acid transport activity from solubilized bovine renal brush-border membrane vesicles was found to react specifically with the enzyme aminopeptidase N. Cleavage of aminopeptidase N from the membranes with papain inhibited Na(+)-dependent amino acid transport activity without affecting that of alpha-methyl D-glucoside. Removal of aminopeptidase substantially increased the Km values for the Na(+)-dependent transport of alanine, glutamine, leucine and phenylalanine without affecting the Vmax. Both Na(+)-dependent amino acid transport and aminopeptidase activity in intact vesicles were competitively inhibited by amino acids with very similar specificity. These results suggest that the amino acid-binding sites of aminopeptidase N and the transporter interact in some way to increase the Km of the transport process for its substrates. However, independent direct inactivation of the transport system by papain cannot be ruled out.

Alanine↗

PubMed2006

Plasma aminopeptidase activities in Parkinson's disease.

Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by a progressive motor disorder, but frequently is accompanied by autonomic symptoms such as hypotension. Together with the decrease of dopamine, significant decreases in aminopeptidase activities have been reported in PD brains. However, up to date there are no studies about changes of aminopeptidase activities in plasma of PD patients. We studied plasma activities of alanyl-, aspartyl-(AspAP), cystinyl-(CysAP) and glutamyl-aminopeptidase (GluAP) in two groups of subjects: control (n=41) and PD (n=48). Plasma activities of AspAP, CysAP, and GluAP showed significant decreases of 24.9% (p<0.05), 39.4% (p<0.01) and 33.3% (p<0.01), respectively, in PD group. These aminopeptidases are involved in the metabolism of circulating peptides such as the ones of the renin-angiotensin system. The importance of aminopeptidases in striatal dopamine content and in neuroendocrine system in PD is discussed.

Aged↗

PubMed1996

Molecular cloning and expression of rat liver aminopeptidase B.

We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6). The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A4 hydrolase. Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-beta-naphthylamidase activity. The recombinant protein was purified to homogeneity from the recombinant E. coli extracts. The enzyme had Cl--dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.

Amino Acid Sequence↗

PubMed2003

Aminopeptidase inhibitors inhibit proliferation and induce apoptosis of K562 and STI571-resistant K562 cell lines through the MAPK and GSK-3beta pathways.

A tyrosine kinase inhibitor, STI571, has been demonstrated to be effective for the treatment of chronic myelogenous leukemia (CML). STI571 inhibits tyrosine kinase activity of ABL and induces apoptosis of CML cells. However, drug resistance develops commonly in patients with blast phase CML, and has become a significant therapeutic problem. We examined the effects of aminopeptidase inhibitors on CML cell line (K562) and a STI571-resistant subline of K562. Ubenimex and the more potent aminopeptidase inhibitor, actinonin, inhibited proliferation of both K562 cells and STI571-resistant K562 cells and also induced their apoptosis in dose- and time-dependent manners. Ubenimex and actinonin induced the activation of caspase-3, and the induction of apoptosis was inhibited by pan-caspase inhibitor, indicating this apoptosis is caspase-dependent. We found that serine phosphorylation of both MAPK and glycogen synthase kinase-3beta were suppressed by aminopeptidase inhibitors in parent K562 and STI571-resistant K562 cells. The expression level of cyclin D1 protein was also reduced by ubenimex and actinonin in both cell lines. These results indicated STI571-resistance does not confer the cross-resistance to aminopeptidase inhibitors in K562 cells and revealed the new findings of aminopeptidase inhibitor-induced intracellular signaling pathways.

Aminopeptidases↗

PubMed1989

Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer.

Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.

Aminopeptidases↗

PubMed2000

Chicken intestinal aminopeptidase: partial sequence of the gene, expression and activity.

Aminopeptidases are members of a membrane-bound metallopeptidase family that are expressed at a high level on the brush-border membrane of enterocytes. Because the rapid growth of meat-type chickens depends on the dietary supply of amino acids, a study of intestinal aminopeptidases, which play a central role in protein digestion, is important. This study is the first reported isolation of the partial cDNA of chicken intestinal aminopeptidase and sequencing of a 1.7-kb cDNA fragment. The gene was isolated by reverse transcriptase polymerase chain reaction using six primers chosen from conserved regions of the aminopeptidase genes. Amplified fragments were extracted from the gel, purified, and sequenced. By using this chicken cDNA as a probe, northern blot analysis revealed a transcript of approximately 3.5 kb in the chicken duodenum, jejunum, and ileum tissues. Higher RNA expression and activity of aminopeptidase were found in the ileum tissue compared with the duodenum and jejunum segments.

Amino Acid Sequence↗

PubMed1993

Aminopeptidases: structure and function.

Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates. They are widely distributed throughout the animal and plant kingdoms and are found in many subcellular organelles, in cytoplasm, and as membrane components. Several aminopeptidases perform essential cellular functions. Many, but not all, of these peptidases are zinc metalloenzymes and are inhibited by the transition-state analog bestatin. Some are monomeric, and others are assemblies of relatively high mass (50 kDa) subunits. cDNA sequences are available for several aminopeptidases, and a 3-dimensional structure is available for the bovine lens enzyme. Crystallographic, electron micrographic, NMR, and photoaffinity labeling studies indicate that lens leucine aminopeptidase protomers are bilobal and that bestatin and substrates are bound in an active site, which is found in the larger lobe on each protomer. Zn2+ is involved in substrate liganding in most aminopeptidases. There is no evidence of an acyl-enzyme intermediate in hydrolysis. Amino acid sequences determined directly or deduced from cDNAs indicate some amino acid sequence homologies in organisms as diverse as Escherichia coli and mammals, particularly in catalytically important residues or in residues involved in metal ion binding.

Amino Acid Sequence↗

PubMed1980

Aminopeptidase activity associated with purified murine leukaemia viruses.

Rauscher leukaemia virus (RLV), extensively purified by rate and density gradient centrifugation procedures, exhibited aminopeptidase (AP) activity. The amount of virion activity was about 0.005 times the specific activity of purified hog kidney aminopeptidase M (EC 3.4.11.2). The activity was also found in purified Moloney and Gross leukaemia viruses, but not in comparable gradient fractions of uninfected JLSV-9 cells. Furthermore replacement of serum by bovine serum albumin in the growth medium of virus producing cells did not affect the AP activity. These results indicate that murine leukaemia viruses (MuLV) possess a tightly bound AP activity which is present as a minor component of the virion preparation and is probably not due to host cell membrane or serum contamination. Characterization of the pH optimum and substrate specificity show the MuLV aminopeptidase activity is similar to but different from both hog kidney, leucine aminopeptidase (EC 3.4.11.1) and aminopeptidase M (EC 3.4.11.2).

AKR murine leukemia virus↗

PubMed2001

Lysine aminopeptidase of Aspergillus niger.

Conserved regions within the M1 family of metallo-aminopeptidases have been used to clone a zinc aminopeptidase from the industrially used fungus Aspergillus niger. The derived amino acid sequence of ApsA is highly similar to two yeast zinc aminopeptidases, LAPI and AAPI (53.3 and 50.9% overall similarity, respectively), two members of the M1 family of metallo-aminopeptidases. The encoding gene was successfully overexpressed in A. niger and the overexpressed product was purified and characterized. Aminopeptidase A was found to be active towards a number of amino acid p-nitroanilide (pNA) substrates, viz. K-pNA, R-pNA, L-pNA, M-pNA, A-pNA and F-pNA. The most preferred N-terminal amino acid is lysine and not leucine, arginine or alanine, the N-terminal amino acids preferred by the yeast homologues. The K(m) and K(cat) for K-pNA and L-pNA were 0.17 mM and 0.49 microkat mg(-1), and 0.16 mM and 0.31 microkat mg(-1), respectively. The pH optimum of the enzyme is between 7.5 and 8, whereas the enzyme is stable between pH 5 and 8. The enzyme is inhibited by the metal chelators EGTA, EDTA and 1,10-phenanthrolin. Bestatin was also able to inhibit the activity.

Aminopeptidases↗

PubMed1995

Characterization of the aminopeptidase activity of epidermal leukotriene A4 hydrolase against the opioid dynorphin fragment 1-7.

Leukotriene A4 hydrolase is a bifunctional cytosolic enzyme, which both hydrolyses leukotriene A4 (LTA4) into leukotriene B4 (LTB4) and exerts aminopeptidase activity against opioid peptides. In the present study we have investigated whether the peptides angiotensin I and II, bradykinin, kallidine, histamine, dynorphin fragment 1-7 and substance P can act as substrates for epidermal and neutrophil LTA4 hydrolase. Among the tested substrates, dynorphin fragment 1-7 was found to be the best substrate for the enzyme. The aminopeptidase activity of epidermal and neutrophil LTA4 hydrolase against dynorphin fragment 1-7 was further characterized. The enzyme was purified from human epidermis and human neutrophils by anion exchange chromatography (Q-Sepharose) and affinity chromatography on a column with the LTA4 hydrolase inhibitor bestatin coupled to AH-Sepharose. The incubation of the dynorphin fragment 1-7 with LTA4 hydrolase resulted in the formation of tyrosine. The presence of the N-terminal amino acid tyrosine is essential for the interaction of opioids with their receptors, and this finding indicates that the LTA4 hydrolase can inactivate dynorphin fragment 1-7. After the two purification steps no other aminopeptidases acting at the N-terminal tyrosine of dynorphin fragment 1-7 was present in the preparation. This was demonstrated by the abolishment of the degradation at the N-terminal end of dynorphin fragment 1-7 when preincubating the enzyme preparation with LTA4 before the incubation with the dynorphin fragment 1-7. The abolishment of the aminopeptidase activity shows that activation of the hydrolase part of the enzyme, with conversion of LTA4 into the potent proinflammatory compound LTB4, results in an inhibition of the aminopeptidase activity of the enzyme. As a result, the catabolism of dynorphin fragment 1-7 and probably of other opioid peptides is inhibited, resulting in sustained biological effects of these opioids. This phenomenon may be important for the maintenance of inflammation in skin conditions, such as psoriasis and atopic dermatitis, in which LTB4 is formed.

Amino Acid Sequence↗

PubMed1982

Structural studies on microvillus aminopeptidase from pig small intestine.

Two amphiphilic forms of intestinal microvillus aminopeptidase were obtained by purification of the enzyme in the absence and in the presence of aprotinin respectively. By subsequent treatment with trypsin in vitro a hydrophilic form was obtained. These three enzyme preparations all contained three subunits. A, B and C (molecular weight 168000, 11800 and 54000 respectively). The individual subunits were purified by gel filtration on Ultrogel AcA 22 in the presence of sodium dodecyl sulphate. Subunits A and B from aminopeptidase prepared in the presence of aprotinin contained both N-terminal lysine and alanine, whereas N-terminal alanine and glycine were found for both of these subunits from the other amphiphilic form. Conversion of amphiphilic aminopeptidase to the hydrophilic form in vitro led only to a change in the N terminus of the B subunit to a valine. N-terminal serine was found in subunit C from all three forms of the aminopeptidase. The amino acid composition and peptide maps of the isolated subunits from one of the amphiphilic forms are reported. It is suggested that subunit B and C arise by cleavage of subunit A, whereas it seemed unlikely that C originates from B. Brush border membrane vesicles were cross-linked by dimethyl-3,3-dithiobispropionimidate, and the aminopeptidase subsequently isolated. Analysis of molecular weights and subunit composition of its constituent polypeptide complexes by two-dimensional polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulphate showed that AB, BBC, BCC, BB, BC and CC complexes had been formed. The subunit structures ABC and BBCC were thus proposed in agreement with the maximum molecular weight of the membrane-bound enzyme previously determined as 330000.U

Amino Acids↗

PubMed1988

Aminopeptidase yscII of yeast. Isolation of mutants and their biochemical and genetic analysis.

Mutant strains of the yeast Saccharomyces cerevisiae defective in aminopeptidase yscII were isolated by screening for reduced external activity against the chromogenic substrate lysine beta-naphthylamide. One of the selected mutant strains analyzed in detail showed wild-type staining activity when tested at 23 degrees C but mutant activity after exposure to 37 degrees C, suggesting a temperature-sensitive mutation. Electrophoretic separation of mutant crude extracts on non-denaturing polyacrylamide gels and subsequent activity staining using lysine and leucine beta-naphthylamides as substrates revealed that in all strains isolated the same distinct activity band was affected, which corresponded to the aminopeptidase activity identified previously as aminopeptidase yscII [Achstetter, T., Ehmann, C. & Wolf, D. H. (1983) Arch. Biochem. Biophys. 226, 292-305]. All mutants strains isolated fell into the same complementation group. Tetrad dissection of sporulated diploids heterozygous for the wild-type and mutant allele resulted in a 2:2 segregation of mutant and wild-type phenotype indicating a single gene mutation. The characteristics of the mutations analyzed point to the gene which we called APE2 as the structural gene of aminopeptidase yscII. No vital consequences of aminopeptidase yscII deficiency on cell life and differentiation could be detected. However, the enzyme seems to be involved in the cellular supply of leucine from externally offered leucine-containing dipeptide substrates.

Aminopeptidases↗

PubMed1991

Aminopeptidase P from rat brain. Purification and action on bioactive peptides.

Aminopeptidase P (EC 3.4.11.9) was purified from rat brain cytosol. A subunit Mr of 71,000 was determined for the reduced, denaturated protein whereas an Mr of 143,000 was determined for the native enzyme. The purified aminopeptidase P selectively liberated all unblocked, preferentially basic or hydrophobic ultimate amino acids from di-, tri- and oligopeptides with N-terminal Xaa-Pro- sequences. Corresponding peptides with penultimate Ala instead of Pro were cleaved with much lower rates; oligopeptides with residues other than Pro or Ala in the penultimate position appeared not to be substrates for the enzyme. Several bioactive peptides with Xaa-Pro sequences, especially bradykinin, substance P, corticortropin-like intermediate lobe peptide, casomorphin and [Tyr]melanostatin were shortened by the N-terminal amino acid by aminopeptidase P action. Rat brain aminopeptidase P was optimally active at pH 7.6-8.0 in the presence of Mn2+. Chelating agents and SH-reacting reagents inhibited the enzyme, but common inhibitors of aminopeptidases, like amastatin or bestatin, of prolidase or of dipeptidyl peptidases II and IV, like N-benzoyloxycarbonyl-proline or epsilon-benzyl-oxycarbonyl-lysyl-proline, as well as antibiotics like beta-lactam ones, bacitracin or puromycin, had little or no effect.

Amino Acid Sequence↗

PubMed1983

An aminopeptidase from mouse brain cytosol that cleaves N-terminal acidic amino acid residues.

An aminopeptidase with specificity directed toward peptides with acidic N-terminal amino acid residues has been isolated from mouse brain cytosol. Purification by ion-exchange chromatography and gel filtration resulted in an enzyme that hydrolyzed aspartyl-phenylalanine methyl ester at a rate of 13.2 mumols/min/mg protein at pH 7.5, an increase in specific activity of 1000-fold over that of brain homogenate. Its apparent molecular weight, determined by gel filtration, is approximately 450,000. Dipeptides with N-terminal aspartyl residues are cleaved preferentially to glutamic-containing analogs, and a neutral amino acid (or histidine) is necessary in the adjacent position. For peptides of the form aspartyl-X, relative activity was 100, 81, 71, 66, 19, or 0, where X was alanine, serine, leucine, phenylalanine, histidine, or proline, respectively. Tripeptides were more rapidly hydrolyzed than dipeptides; however, activity tended to decline with increasing chain length. The acidic aminopeptidase can account for almost all of the activity of brain cytosol toward the N-terminal aspartyl residue of angiotensin II, aspartyl-phenylalanine methyl ester or aspartyl-alanine, and the N-terminal glutamyl residue of adrenocorticotropin(5-10). The enzyme was unaffected by bestatin or amastatin. It was inhibited by o-phenanthroline and EDTA. The latter effect could be reversed completely by Zn2+ and partially by Mn2+ or Mg2+; Co2+ and Fe2+ had no effect; Ca2+ was inhibitory. These properties distinguish the brain acidic aminopeptidase from aminopeptidase A isolated from human serum or pig kidney and the aspartyl aminopeptidase of dog kidney.

Amino Acids↗

PubMed1996

Regulation of the yeast vacuolar aminopeptidase Y gene (APY1) expression.

The steady-state levels of the aminopeptidase Y (APY1) transcript and the level of assayable aminopeptidase Y activity were measured under a variety of nutritional conditions. Specific APY1 mRNA is less abundant in cells grown in minimal medium than in cells grown in rich medium, while active enzyme levels follow just the opposite pattern. Aminopeptidase Y activity decreases when cells are deprived of glucose without a concomitant fall in mRNA levels. Production of aminopeptidase Y is not markedly affected by nitrogen regulation. APY1 gene expression is not disturbed in heat-shocked cells. Our data support the idea that the main control event in vacuolar aminopeptidase Y expression is a post-transcriptional step.

Aminopeptidases↗