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PubMed1986

Purification and characterization of ascamycin-hydrolysing aminopeptidase from Xanthomonas citri.

A nucleoside antibiotic, ascamycin (9-beta-[5'-0-(N-L-alanyl) sulphamoyl-D-ribofuranosyl]-2-chloroadenine), has a selective antibacterial activity against Xanthomonas species. When ascamycin was dealanylated, dealanylascamycin showed a broad antibacterial activity against various Gram-negative and Gram-positive bacteria. Xanthomonas citri is susceptible to ascamycin by virtue of the ascamycin-dealanylating enzyme on the cell surface [Osada & Isono (1985) Antimicrob. Agents Chemother. 27, 230-233]. The enzyme (Xc aminopeptidase) was purified from X. citri cells by successive DEAE-cellulose, chromatofocusing and Sephadex G-100 column chromatography to a homogeneous state. The purified enzyme exhibited a single band with an Mr of 38 000 in SDS/polyacrylamide-gel electrophoresis. Gel filtration on a calibrated column indicated a similar Mr value. The isoelectric point of the enzyme was 5.7. The enzyme catalysed the hydrolysis of the alanyl group of ascamycin and liberated alanine from the sulphamoyl nucleoside. The enzyme also catalysed the hydrolysis of L-proline beta-naphthylamide and L-alanine beta-naphthylamide. The optimal pH and temperature for enzyme activity were pH 7.5-8.0 and 35-40 degrees C respectively. The enzyme was inhibited by thiol-enzyme inhibitors (i.e. rho-chloromercuribenzoate and N-ethylmaleimide), but was not affected by various naturally occurring aminopeptidase inhibitors (i.e. amastatin, bestatin, pepstatin and leupeptin). Mn2+ and Mg2+ activated the enzyme, whereas Cu2+, Zn2+ and Cd2+ were inhibitory.

2-Chloroadenosine↗
PubMed1987

An improved spectrophotometric assay for leucine aminopeptidase.

A sensitive assay to determine the activity of leucine aminopeptidase (EC 3.4.11.1), using L-leucine thiobenzyl ester as substrate, was developed. Hydrolysis of the ester by leucine aminopeptidase can be monitored in the presence of 5,5-dithiobis-(2-nitrobenzoic acid) by continuous spectrophotometric measurement at 412 nm. Comparison with some amide substrates showed that the thiol ester provides a much more sensitive assay, its specificity constant (Vmax./Km) being some 3000-fold higher than that of leucine p-nitroanilide.

Animals↗
PubMed1987

Proteins of the kidney microvillar membrane. Enzymic and molecular properties of aminopeptidase W.

Aminopeptidase W is a newly discovered enzyme of the renal and intestinal brush borders, having been first isolated as a 130 kDa glycoprotein recognized by a monoclonal antibody [Gee & Kenny (1985) Biochem. J. 230, 753-764]. It is particularly effective in the hydrolysis of dipeptides, Glu-Trp (Km 0.57 mM; kcat. 6770 min-1) being a favoured substrate. Dipeptides with tryptophan, phenylalanine or tyrosine in the P1 position were rapidly hydrolysed, but the requirements in respect of the P1 residue were not stringent. The activity of aminopeptidase W is markedly influenced by ionic conditions. The highest activity was observed in 100 mM-Tris/HCl, pH 8; phosphate ions were strongly inhibitory. Activity was also greatly affected by bivalent metal ions, and the magnitude and direction of the effects depended on the nature of the buffer anions and on pH. The most effective inhibitors were amastatin and bestatin. Some thiols also inhibited, but other chelating agents, EDTA and 1,10-phenanthroline, had no effect over the concentration range 1-10 mM. Other group-specific inhibitors, for cysteine, serine or aspartic peptidases, were also ineffective. Some molecular properties were studied. Deglycosylation by treatment with N-glycanase diminished the apparent subunit Mr from 130,000 to 90,000. The enzyme contained zinc, 1.2 atoms/subunit, and in spite of the atypical properties of this enzyme in respect of chelating agents, a zinc-catalysed mechanism is the most probable. Its roles in digestion and in renal function are not yet clear.

Aminopeptidases↗
PubMed1996

Molecular cloning and expression in COS-1 cells of pig kidney aminopeptidase P.

Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9), a key enzyme in the metabolism of the vasodilator bradykinin, has been cloned from a pig kidney cortex cDNA library following the use of the PCR to identify sub-libraries enriched in AP-P clones. The complete primary sequence of the enzyme has been deduced from a full-length cDNA clone. This predicts a protein of 673 amino acids with a cleavable N-terminal signal sequence and six potential N-linked glycosylation sites. A stretch of mainly hydrophobic amino acids at the C-terminus is predicted to co-ordinate the attachment of a glycosyl-phosphatidylinositol (GPI) membrane anchor. Although AP-P is a zinc metallopeptidase, the predicted primary sequence does not contain any recognizable zinc-binding motif. Transient expression of AP-P cDNA in COS-1 cells resulted in enzymic activity characteristic of AP-P, namely apstatin- and EDTA-sensitive hydrolysis of bradykinin and Gly-Pro-Hyp. The expressed protein was recognized as a polypeptide of M(r)91,000 under reducing conditions, following immunoblotting of COS-1 membranes with a polyclonal antibody raised against purified pig kidney AP-P. The presence of a GPI anchor on expressed AP-P was established by demonstrating release of the enzyme from a membrane fraction following treatment with bacterial phosphatidylinositol-specific phospholipase C and its corresponding conversion from an amphipathic to a hydrophilic form, as assessed by phase separation in Triton X-114. Sequence comparisons confirm that AP-P is a member of the proline peptidase family of hydrolytic enzymes and is unrelated in sequence to other brush-border membrane peptidases.

Amino Acid Sequence↗
PubMed2001

Functional expression and characterization of the cytoplasmic aminopeptidase P of Caenorhabditis elegans.

Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9) cleaves the N-terminal X-Pro bond of peptides and occurs in mammals as both cytosolic and plasma membrane forms, encoded by separate genes. In mammals, the plasma membrane AP-P can function as a kininase, but little is known about the physiological role of the cytosolic enzyme. The C. elegans genome contains a single gene encoding AP-P (W03G9.4), analysis of which predicts regions displaying high levels of amino-acid sequence homology between the predicted gene product and mammalian cytoplasmic AP-P, with the absolute conservation of key catalytic residues. The sequence of an EST (yk91g4), comprising the open reading frame of W03G9.4, confirmed the predicted genomic structure of the gene and the prediction that W03G9.4 codes for a nonsecreted protein with a molecular mass of 68 kDa. Nematodes transformed with a promoter reporter construct, W03G9.4:GFP, showed high levels of fluorescence in the intestine of larvae and adult hermaphrodites, indicating that the intestine is a major site of W03G9.4 expression. yk91g4 tagged with a hexahistidine and DLYDDDDK peptide epitope was expressed in Escherichia coli to yield, after affinity purification, a recombinant protein with a molecular mass of 71 kDa. The recombinant W03G9.4 removed the N-terminal amino acid from bradykinin (RPPGFSPFR), a Caenorhabditis elegans neuropeptide (KPSFVRFamide) and Lem Trp 1 (APSGFLGVRamide), but did not display activity towards angiotensin I (NRVYIHPFHL), des-Arg bradykinin and AF1 (KNEFIRFamide). The activity towards bradykinin was inhibited by EDTA and 1, 10 phenanthroline, as expected for a metalloenzyme, and also by apstatin (IC50, 1 microM), a selective inhibitor of mammalian AP-P. A Km of 45 microM and an optimum pH of 7-8 was observed with bradykinin as the substrate. The activity of the nematode AP-P, like its mammalian counterparts, was strongly influenced by metal ions, with Co2+, Mn2+ and Zn2+ all inhibiting the hydrolysis of bradykinin. We conclude that W03G9.4 codes for a cytoplasmic AP-P with very similar enzymatic properties to those of mammalian AP-P, and we suggest that the enzyme has a physiological role in the intracellular hydrolysis of proline-containing peptides absorbed from the lumen of the intestine.

Amino Acid Sequence↗
PubMed1999

Overexpression, purification and biochemical characterization of the wound-induced leucine aminopeptidase of tomato.

Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A). While the expression of LapA genes are well characterized, the specificity of the LAP-A enzyme has not been studied. The LAP-A preprotein and mature polypeptide were overexpressed in Escherichia coli. PreLAP-A was not processed and was inactive accumulating in inclusion bodies. In contrast, 55-kDa mature LAP-A subunits assembled into an active, 357-kDa enzyme in E. coli. LAP-A from E. coli cultures was purified to apparent homogeneity and characterized relative to its animal (porcine LAP) and prokaryotic (E. coli PepA) homologues. Similar to the porcine and E. coli enzymes, the tomato LAP-A had high temperature and pH optima. Mn2+ was a strong activator for all three enzymes, while chelators, zinc ion, and the slow-binding aminopeptidase inhibitors (amastatin and bestatin) strongly inhibited activities of all three LAPs. The substrate specificities of porcine, E. coli and tomato LAPs were determined using amino-acid-p-nitroanilide and -beta-naphthylamide substrates. The tomato LAP-A preferentially hydrolyzed substrates with N-terminal Leu, Met and Arg residues. LAP-A had substantially lower levels of activity on other chromogenic substrates. Several differences in substrate specificities for the animal, plant and prokaryotic enzymes were noted.

Animals↗
PubMed1997

Nociceptin/orphanin FQ metabolism: role of aminopeptidase and endopeptidase 24.15.

The endogenous opioid receptor-like1 (ORL1) ligand, nociceptin/orphanin FQ (FGGFTGARKSARKLANQ), a heptadecapeptide structurally resembling dynorphin A, has recently been identified. The wide distribution of ORL1 mRNA and nociceptin/orphanin FQ precursor in the CNS, particularly in the limbic system regions and in several areas known to be involved in pain perception, suggests that nociceptin/orphanin FQ is potentially endowed with various central functions. In general, activation and/or inactivation of regulatory peptides occur through the action of cell surface peptidases. The physiological mechanisms under which nociceptin/orphanin FQ is metabolized should lead to a better understanding of its physiological functions. Mouse brain cortical slices were incubated in medium containing the heptadecapeptide in the presence or in the absence of peptidase inhibitors. The critical sites of enzymatic cleavage are Phe1-Gly2, Ala7-Arg8, Ala11-Arg12, and Arg12-Lys13 bonds. The major role played by metallopeptidases was confirmed by the complete protection of metabolism in the presence of EDTA. Aminopeptidase N and endopeptidase 24.15 are the two main enzymes involved in nociceptin/orphanin FQ metabolism, whereas endopeptidase 24.11 (involved in enkephalin [YGGFM(L)] catabolism) does not appear critically involved in nociceptin/orphanin FQ metabolism. The physiological relevance of aminopeptidase N and endopeptidase 24.15 in the heptadecapeptide metabolism remains to be determined.

Aminopeptidases↗
PubMed1997

Cloning of the human puromycin-sensitive aminopeptidase and evidence for expression in neurons.

The puromycin-sensitive aminopeptidase (PSA) is thought to contribute to the degradation of enkephalins. Besides being the most abundant aminopeptidase in the brain, PSA is expressed in other organs as well. From a human fetal brain cDNA library, we have isolated a cDNA encoding the human PSA (huPSA) protein. The isolated cDNA gave rise to a protein with a molecular mass of 99 kDa. Compared with mouse PSA, homology at the amino acid and cDNA level was 98 and 93%, respectively. Translation of the huPSA was found to be initiated at the second of two possible start codons, as shown by studies with antibodies directed against peptide sequences of both potential N-terminal regions. Northern blot analysis with RNA isolated from different human organs demonstrated that the huPSA transcript is strongest but not exclusively expressed in the brain. Vesicular stomatitis virus epitope-tagged huPSA protein was expressed in HeLa cells and found to be localized in the cytoplasm, especially in the perinuclear region. By in situ hybridization, huPSA transcript could be identified in cortical and cerebellar neurons, whereas glial cells and blood vessels remained negative.

Amino Acid Sequence↗
PubMed1997

Isolation and characterization of proteinase- and aminopeptidase-deficient mutants of Lactobacillus casei subsp. casei IFPL 731.

Proteinase-deficient (Prt-) and aminopeptidase-deficient (Amp-) variants of Lactobacillus casei subsp. casei IFPL 731 were isolated and characterized. The Prt- mutant was isolated from strains that developed poorly on glucose milk agar. The Amp- mutant was isolated on the basis of its inability to hydrolyse L-leucine-beta-naphtylamide. The Prt- variant developed poorly, while in milk the Amp- variant grew at about the same rate as the parental strain. The characterization of aminopeptidase activity in more detail showed that at least two enzymes are involved The results of the present study suggest that the proteolytic system of Lactobacillus casei is subjected to a regulatory system.

Aminopeptidases↗
PubMedDate not supplied

Aminopeptidase of Eimeria nieschulzi physico-chemical properties and action of antimalarial drugs.

An aminopeptidase capable of degrading synthetic substrates and short peptides has been demonstrated in Eimeria nieschulzi sporulated oocysts. Physico-chemical properties and the classic activator and inhibitory effects have been determined. The inhibitory activity of classic antimalarial drugs has been demonstrated and the study of the inhibitory mechanism by Chloroquine has been further studied. Lastly, the comparison between the aminopeptidase of Eimeria genus parasites and that of Plasmodium has been discussed.

Aminopeptidases↗
PubMed1994

Cadmium action on aminopeptidase N activity and L-threonine intestinal transport in rabbit.

Cadmium has been recognized as an environmental contaminant. In oral cadmium intoxication, the immediate target organ is the gastrointestinal tract. The aim of the present work was to study the effect of cadmium on the intestinal absorption of L-threonine and on the aminopeptidase N activity in rabbit jejunum, after in vitro addition and/or oral administration of CdCl2 in drinking water. Results obtained show that cadmium decreases L-threonine absorption in the jejunal tissue. This effect seems to be due to an action mainly on active amino-acid transport at the mucosal border of the intestinal epithelium, because cadmium does not seem to modify the amino-acid diffusion across the intestinal epithelium. Cadmium also inhibits the (Na(+)-K+)-ATPase activity of the enterocyte, which might explain the inhibition of the Na(+)-dependent L-threonine transport. Nevertheless, a direct action of the cadmium ion on the carrier of active transport cannot be rejected. Cadmium modifies the aminopeptidase N activity when administered in drinking water for 4 d.

Adenosine Triphosphatases↗
PubMed1987

Effect of microsomal leucine aminopeptidase from human placenta (microsomal P-LAP) on pressor response to infused angiotensin II (A-II) in rat.

The role of microsomal placental leucine aminopeptidase (microsomal P-LAP) in the decreased pressor responsiveness to angiotensin II (A-II) in pregnancy was studied. Appreciable amounts of microsomal P-LAP activity were found in rat placenta. The similar dose to the endogenous activity, of human microsomal P-LAP exogenously administered to rats, resulted in significant decrease in the response to A-II. Bestatin, an inhibitor of the microsomal leucine aminopeptidase administered to pregnant rats, enhanced the A-II response. Therefore our present study suggests such refractoriness in response to A-II in pregnancy is due to increased inactivation by the microsomal P-LAP. It was also suggested that prostaglandins were not involved in such refractoriness by the experiments with indomethacin.

Angiotensin II↗
PubMed1988

In vitro degradation of opioid peptides by human placental aminopeptidase M.

The degradation of opioid peptides by human placental aminopeptidase M was studied by measuring liberated amino acids by high performance liquid chromatography. The purified placental aminopeptidase M actively degraded Met-enkephalin (Met-Enk) and Leu-enkephalin (Leu-Enk) completely into constituent 5 amino acids. The activities as a function of tyrosine liberation were observed for Met-Enk followed by Leu-Enk, beta-neoendorphin, dynorphin, and beta-endorphin.

Aminopeptidases↗
PubMed1988

Relationship between endo- and exopeptidases in a processing enzyme system: activation of an endoprotease by the aminopeptidase B-like activity in somatostatin-28 convertase.

The somatostatin-28 convertase activity involved in vitro in the processing of somatostatin-28 into the neuropeptides somatostatin-28-(1-12) and somatostatin-14 is composed of an endoprotease and a basic aminopeptidase. We report herein on the purification to apparent homogeneity of these two constituents and on their functional interrelationship. In particular we observed that after various physicochemical treatments, the 90-kDa endoprotease activity was recovered both at this molecular mass and as a 45-kDa entity. Moreover, the production of [Arg-2,Lys-1]somatostatin-14 from somatostatin-28 by the action of the endoprotease was activated in a cooperative manner by the aminopeptidase B-like enzyme. A 10-fold activation occurred when the exopeptidase was inhibited by 6.5 mM diisopropyl fluorophosphate and allowed the determination of a half-maximal activation constant (K1/2) of approximately equal to 13 nM. These observations strongly suggest that both enzymes act in a concerted manner in vitro and that they may form a complex in vivo.

Aminopeptidases↗
PubMed1997

Aminopeptidase B from the rat testis is a bifunctional enzyme structurally related to leukotriene-A4 hydrolase.

An aminopeptidase B (Ap-B) was previously purified to homogeneity from rat testis extracts and characterized. In the present work, by using oligonucleotides selected on the basis of partial amino acid microsequences of pure Ap-B and PCR techniques, the nucleotide sequence of a 2.2-kb cDNA was obtained. The deduced amino acid sequence corresponds to a 648-residue protein (72.3 kDa) containing the canonical "HEXXHX18E" signature, which allowed its classification as a member of the M1 family of metallopeptidases. It exhibits 33% identity and 48% similarity with leukotriene-A4 hydrolase, a relation further supported by the capacity of Ap-B to hydrolyze leukotriene A4. Both enzymes also were closely related to a partially sequenced protein from Dictyostelium discoideum, which might constitute the putative common ancestor of either aminopeptidase or epoxide hydrolase, or both. Ap-B and its mRNA were detected in the germ line and in the Sertoli and peritubular cells of the seminiferous tubules. Because the enzyme was found in the medium conditioned by spermatocytes and spermatids and in the acrosome during spermatozoa formation, together these observations suggested an involvement of this exometallopeptidase in the secretory pathway. It is concluded that this ubiquitous enzyme may be involved in multiple processing mechanisms.

Amino Acid Sequence↗
PubMed1998

Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli.

The structure of the proline-specific aminopeptidase (EC 3.4.11.9) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-A resolution, for a dipeptide-inhibited complex at 2.3-A resolution, and for a low-pH inactive form at 2.7-A resolution. The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions. The monomer folds into two domains. The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro. The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits. The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme). The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.

Aminopeptidases↗
PubMed2002

Methionine aminopeptidase 2 is a new target for the metastasis-associated protein, S100A4.

S100A4 is an EF-hand type calcium-binding protein that regulates tumor metastasis and a variety of cellular processes via interaction with different target proteins. Here we report that S100A4 physically interacts with methionine aminopeptidase 2 (MetAP2), the primary target for potent angiogenesis inhibitors, fumagillin and ovalicin. Using a yeast two-hybrid screen, S100A4 was found to interact with the N-terminal half of MetAP2. In vitro pull-down assays showed that S100A4 associates with MetAP2 in a calcium-dependent manner. In addition, the binding site of S100A4 was found located within the region between amino acid residues 170 and 229 of MetAP2. In vivo interaction of S100A4 with MetAP2 was verified by co-immunoprecipitation analysis. Immunofluorescent staining revealed that S100A4 and MetAP2 were co-localized in both quiescent and basic fibroblast growth factor-treated murine endothelial MSS31 cells, in the latter of which a significant change of intracellular distribution of both proteins was observed. Although the binding of S100A4 did not affect the in vitro methionine aminopeptidase activity of MetAP2, the cytochemical observation suggests a possible involvement of S100A4 in the regulation of MetAP2 activity through changing its localization, thereby modulating the N-terminal methionine processing of nascent substrates. These results may offer an essential clue for understanding the functional role of S100A4 in regulating endothelial cell growth and tumor metastasis.

Amino Acid Sequence↗