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At least 19 recordsLinked to original sources
PubMed1990

Comparison of aminopeptidase, dipeptidyl aminopeptidase and tripeptidyl aminopeptidase activities in brain tissue from normal and Alzheimer's disease cases.

Recent evidence has suggested that Alzheimer's disease may result from an underlying defect of protein catabolism. In an attempt to identify such a defect, we have undertaken the systematic identification and characterization of the proteolytic enzymes responsible in human brain for oligopeptide breakdown via hydrolysis of NH2-terminal residues--the aminopeptidases, dipeptidyl aminopeptidases and tripeptidyl aminopeptidases. The activities of these enzymes were determined following anion exchange chromatographic fractionation of cortical soluble extracts, to prevent interference in the respective assay procedures by the major cortical aminopeptidase. Corresponding enzyme types showed similar levels of activity in soluble extracts of frontal cortex from normal and Alzheimer's disease cases. These data suggest that the characteristic neurodegeneration associated with Alzheimer's disease does not result from altered aminopeptidase, dipeptidyl aminopeptidase or tripeptidyl aminopeptidase activity in cortical tissues of patients with this disorder.

Aged↗
PubMed1987

Human skeletal muscle contains two major aminopeptidases: an anion-activated aminopeptidase B and an aminopeptidase M-like enzyme.

Two major aminopeptidases, an aminopeptidase B and an aminopeptidase M-like enzyme, were purified from human skeletal muscle by DEAE-cellulose, HPLC gel filtration, and hydroxyapatite column chromatographies. The purified aminopeptidase B exhibits a molecular weight of 76,000 under both native and denaturing conditions. The activity of the aminopeptidase B is regulated by C1 ions and other anions in vitro. On the other hand, the aminopeptidase M-like enzyme is a monomeric protein having a molecular weight of 96,000. It is capable of significantly cleaving Phe-, Leu-, Arg-, and Ala-aminoacyl bonds in the presence of 2-mercaptoethanol. The pH optima for both enzymes are around 7.0, and bestatin is an effective inhibitor of both enzymes.

Amino Acids↗
PubMed1989

[A method for the determination of cytosolic aminopeptidase in serum and its usefulness for clinical application. Selection of inhibitors for membrane-bound aminopeptidase and cystyl aminopeptidase].

A method for the determination of cytosolic aminopeptidase (EC 3.4.11.1; C-AP) in serum was developed. At first, more specific and adequate inhibitor for other serum peptidases, such as membrane-bound aminopeptidase (EC 3.4.11.2; arylamidase, AA) and cystyl aminopeptidase (EC 3.4.11.3; CAP) was selected from 1,10-phenanthroline derivatives. The compound, 4,7-dimethyl-1,10-phenanthroline (4,7-DMP) is one of the most effective inhibitor for AA and CAP, and it inhibits completely these enzymes at the concentration of less than 0.4 mmol/l. C-AP in serum at an optimum pH of 8.0 in the assay using L-leucine amide (LA) as the substrate had no inhibitory effects at the concentration of more than 0.4 mmol/l of 4,7-DMP. The results with the proposed method correlated with those with a conventional electrophoretic method. The proposed method hence is a specific and easily available assay for C-AP in serum. The further analysis of C-AP using this method would promise the establishment of clinical assessment of the enzyme.

Aminopeptidases↗
PubMed1986

Purification and characterization of human placental microsomal aminopeptidase: immunological difference between placental microsomal aminopeptidase and pregnancy serum cystyl-aminopeptidase.

Human placental microsomal aminopeptidase (microsomal PAP) was purified 3,880-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100 and also trypsin digestion, and subjected to zinc sulfate fractionation, chromatographies with DE-52, hydroxylapatite, Sephacryl S-300 and lentil lectin-Sepharose 4B, and finally affinity chromatography with bestatin-Sepharose 4B. Microsomal PAP was separated from aminopeptidase A (AAP) by affinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 220,000 by high-performance liquid chromatography with an aqueous gel column. The purified enzyme gave almost a single band with a molecular mass of 140,000 by sodium dodecyl sulfate (SDS) gel electrophoresis. The isoelectric point of the enzyme was 5.2. The purified enzyme was most active at pH 8.0 with L-leucine-p-nitroanilide as substrate; the Km value for this substrate was 1.1 mmol/l. The microsomal PAP was immunologically different from the pregnancy serum cystyl aminopeptidase (serum PAP).

Aminopeptidases↗
PubMed2006

Angiotensin AT4 receptor ligand interaction with cystinyl aminopeptidase and aminopeptidase N: [125I]Angiotensin IV only binds to the cystinyl aminopeptidase apo-enzyme.

Due to its high affinity for [(125)I]Angiotensin IV, cystinyl aminopeptidase (CAP) has recently been assigned as the 'angiotensin AT(4) receptor'. Since the aminopeptidase N (AP-N) activity is also susceptible to inhibition by Angiotensin IV, it might represent an additional target for this peptide. Based on [(125)I]Angiotensin IV binding and catalytic activity measurements, we compared the ligand interaction properties of recombinant human CAP and human AP-N. Both enzymes displayed distinct pharmacological profiles. Although their activity is inhibited by Angiotensin IV and LVV-hemorphin 7, both peptides are more potent CAP-inhibitors. On the other hand, substance P and l-methionine have a higher potency for AP-N. High affinity binding of [(125)I]Angiotensin IV to CAP occurs in the presence of chelators but not to AP-N in either the absence or presence of chelators. These differences were exploited to determine whether CAP and/or AP-N are present in different cell lines (CHO-K1, COS-7, HEK293, SK-N-MC and MDBK). We provide evidence that CAP predominates in these cell lines and that, comparatively, CHO-K1 cells display the highest level of this enzyme.

Angiotensin II↗
PubMed1999

Dipeptidyl aminopeptidase IV and aminopeptidase P, two proline specific enzymes from the cytoplasm of guinea-pig brain: their role in metabolism of peptides containing consecutive prolines.

In this study the majority of dipeptidyl aminopeptidase IV and aminopeptidase P activities of guinea-pig brain are reported to reside in the cytoplasm. Both activities were purified and soluble dipeptidyl aminopeptidase IV was found to have a relative molecular mass of 194000 and to be comprised of two equal subunits of relative molecular mass 93000 while native soluble aminopeptidase P had a relative molecular mass of 140000. Both activities require proline or alanine in the penultimate position from the N-terminus. Dipeptidyl aminopeptidase IV removed the N-terminal dipeptide whereas aminopeptidase P removed only the N-terminal amino acid. Dipeptidyl aminopeptidase IV was inactive if proline was also present in the third position from the N-terminus whereas aminopeptidase P was unable to remove the N-terminal glycyl, pyroglutamyl or prolyl residues even though proline was present in the second position. Soluble dipeptidyl aminopeptidase IV was differentiated from the previously reported particulate form by its sensitivity to p-chloromercuribenzoate, N-ethyl maleimide and puromycin. The metabolism of Leu-Pro-Pro-Ser by guinea-pig cytoplasm was investigated in the presence of inhibitors to evaluate the contribution by dipeptidyl aminopeptidase IV and aminopeptidase P to the hydrolysis of a peptide containing two consecutive proline residues. The results indicated that either dipeptidyl aminopeptidase IV or prolyl oligopeptidase were required along with aminopeptidase P and prolidase to achieve complete hydrolysis of this tetrapeptide.

Aminopeptidases↗
PubMed1991

Structural and immunological evidence for the identity of prolyl aminopeptidase with leucyl aminopeptidase.

Prolyl aminopeptidase (EC 3.4.11.5) has been assumed to be a unique enzyme catalyzing specifically the removal of unsubstituted NH2-terminal L-prolyl residues from various peptides and to be distinct from leucyl aminopeptidase (EC 3.4.11.1). In the present study, prolyl aminopeptidases were purified to apparent homogeneity from pig small intestine mucosa and human liver and their NH2-terminal amino acid sequences were determined together with that of pig kidney leucyl aminopeptidase. The NH2-terminal 24-residue sequence of pig intestinal prolyl aminopeptidase was shown to be identical with that of pig kidney leucyl aminopeptidase. The NH2-terminal sequence of human liver prolyl aminopeptidase was also shown to be very similar to that of pig kidney leucyl aminopeptidase. Further, pig intestinal prolyl aminopeptidase and pig kidney leucyl aminopeptidase were immunologically indistinguishable. These lines of evidence strongly suggest that prolyl aminopeptidase is identical with leucyl aminopeptidase.

Amino Acid Sequence↗
PubMed1983

Proteolysis in eucaryotic cells: aminopeptidases and dipeptidyl aminopeptidases of yeast revisited.

Using nine different L-aminoacyl-4-nitroanilides and four different dipeptidyl-4-nitroanilides, aminopeptidases and dipeptidyl aminopeptidases active at pH 7.5 and (or) pH 5.5 in logarithmically growing and stationary-phase cells of Saccharomyces cerevisiae were searched for. Ion-exchange chromatography was used to separate the proteins of the soluble cell extract. Besides the three already-characterized aminopeptidases--aminopeptidase I (P. Matile, A. Wiemken, and W. Guyer (1971) Planta (Berlin) 96, 43-53; J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta 527, 31-41), aminopeptidase II (J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta 527, 31-41; J. Knüver (1982) Thesis, Fachbereich Chemie, Marburg, FRG), and aminopeptidase Co (T. Achstetter, C. Ehmann, and D. H. Wolf (1982) Biochem. Biophys. Res. Commun. 109, 341-347)--12 additional aminopeptidase activities are found in soluble cell extracts eluting from the ion-exchange column. These activities differ from the characterized aminopeptidases in one or more of the parameters such as charge, size, substrate specificity, inhibition pattern, pH optimum for activity and regulation. Also, a particulate aminopeptidase, called aminopeptidase P, is found in the nonsoluble fraction of disintegrated cells. Besides the described particulate X-prolyl-dipeptidyl aminopeptidase (M. P. Suarez Rendueles, J. Schwencke, N. Garcia-Alvarez and S. Gascon (1981) FEBS Lett. 131, 296-300), three additional dipeptidyl aminopeptidase activities of different substrate specificities are found in the soluble extract.

Amino Acid Sequence↗
PubMed1983

Distribution and biosynthesis of aminopeptidase N and dipeptidyl aminopeptidase IV in rat small intestine.

The regional, cellular and subcellular distribution patterns of aminopeptidase N and dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidase N of brush border membrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidase IV had a more uniform distribution profile with relatively high activity in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cells. Both enzymes were primarily associated with brush border membranes in all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity/mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fold activation of precursor enzyme forms occurs during transfer from the Golgi membranes to the brush border membranes.

Aminopeptidases↗
PubMed1985

Characterization of membrane-bound aminopeptidases from rat brain: identification of the enkephalin-degrading aminopeptidase.

Rat brain aminopeptidase activity was solubilized from membranes by incubation with thiols. This novel procedure resulted in the release of the same two aminopeptidases (MI and MII) previously shown to be solubilized by the nonionic detergent Triton X-100. The solubilized aminopeptidases MI and MII were resolved by ion-exchange chromatography and further purified by hydroxylapatite chromatography. Aminopeptidase MI was shown to hydrolyze only the beta-naphthylamides of arginine and lysine whereas aminopeptidase MII exhibited a broad specificity with respect to amino acid beta-naphthylamides. Only aminopeptidase MII hydrolyzed Leu-enkephalin at a significant rate, indicating that this enzyme can account for the membrane-bound enkephalin aminopeptidase activity. The enkephalin-degrading aminopeptidase is potently inhibited by opioid (alpha-neo-endorphin and dynorphin) as well as nonopioid (substance P, somatostatin, and angiotensin I) peptides in the range of 0.2-2.0 microM. The regional distribution of aminopeptidases MI and MII in rat brain are rather different, with aminopeptidase MII distribution more closely paralleling the distribution of opiate receptors.

Aminopeptidases↗
PubMed1986

Characterization of aminopeptidases responsible for inactivating endogenous (Met5)enkephalin in brain slices using peptidase inhibitors and anti-aminopeptidase M antibodies.

In addition to "enkephalinase" (EC 3.4.24.11), two enkephalin-hydrolyzing aminopeptidases recently identified in cerebral membranes--aminopeptidase M (EC 3.4.11.2) and a "puromycin-sensitive" aminopeptidase (also designated "MII" or "aminoenkephalinase")--are potentially involved in endogenous enkephalin inactivation. Their participation in the hydrolysis of the endogenous (Met5)enkephalin released by depolarization of slices from rat globus pallidus was assessed, using three inhibitory agents: bestatin, puromycin, and anti-aminopeptidase M antibodies. The selectivity and potency of these agents were first determined by evaluating their IC50 values for inhibition of [3H](Met5)enkephalin hydrolysis by increasingly complex preparations comprising semipurified aminopeptidases, pallidal membranes, and pallidal slices. Bestatin was a fairly potent inhibitor but lacked selectivity, as there was only a 3-fold difference between its IC50 values for the two aminopeptidases, and it displayed restricted diffusion and degradation in the slice preparation. Puromycin discriminated well between the two aminopeptidases (30-fold difference in IC50 values) and did not show any apparent restricted diffusion in the slice preparation. Antiaminopeptidase M antibodies were highly discriminant (greater than 300-fold difference in IC50 values for the two aminopeptidases) but displayed restricted diffusion. Analysis of the concentration-protection curves of the three agents for recovery of the (Met5)enkephalin released from pallidal slices in the presence of the "enkephalinase" inhibitor, thiorphan, indicated that both aminopeptidases participated in enkephalin degradation but that the role of aminopeptidase M was largely predominant, in contrast with its low relative activity in the preparation.

Aminopeptidases↗
PubMed1985

Dipeptidyl-aminopeptidases and aminopeptidases in Dictyostelium discoideum.

Extracts prepared from culminating cells of Dictyostelium discoideum have been found to contain dipeptidyl-aminopeptidases I (EC 3.4.14.1), II (EC 3.4.14.2), III (EC 3.4.14.4), arginine aminopeptidase (EC 3.4.11.6) and valine aminopeptidase. Dipeptidyl-aminopeptidase III was the most active of the dipeptidyl-aminopeptidases; its molecular weight was 158,000, with a pH optimum of 10.2 and gave a single peak of activity on gel-filtration or when fractionated by chromatofocusing. The specific activities of dipeptidyl-aminopeptidases I and III increased during development being highest during the culmination stage before decreasing during sorocarp formation; dipeptidyl-aminopeptidase II and arginine aminopeptidase decreased progressively throughout development. The presence of these dipeptidyl-aminopeptidases suggests the possibility that processing of peptides may be necessary during the development of Dictyostelium.

Aminopeptidases↗
PubMed2001

A secreted aminopeptidase of Pseudomonas aeruginosa. Identification, primary structure, and relationship to other aminopeptidases.

Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains. The aminopeptidase was partially purified by DEAE-cellulose chromatography and found to be heat stable. The apparent molecular mass of the enzyme was approximately 56 kDa; hence, it was designated AP(56). Heating (70 degrees C) of the partially purified aminopeptidase preparations led to the conversion of AP(56) to a approximately 28-kDa protein (AP(28)) that retained enzyme activity, a reaction that depended on elastase (LasB). The pH optimum for Leu-pNA hydrolysis by AP(28) was 8.5. This activity was inhibited by Zn chelators but not by inhibitors of serine- or thiol-proteases, suggesting that AP(28) is a Zn-dependent enzyme. Of several amino acid p-nitroanilide derivatives examined, Leu-pNA was the preferred substrate. The sequences of the first 20 residues of AP(56) and AP(28) were determined. A search of the P. aeruginosa genomic data base revealed a perfect match of these sequences with positions 39-58 and 273-291, respectively, in a 536-amino acid residue open reading frame predicted to encode an aminopeptidase. A search for sequence similarities with other proteins revealed 52% identity with Streptomyces griseus aminopeptidase, approximately 35% identity with Saccharomyces cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacillus subtilis, and 29-32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases. The residues potentially involved in zinc coordination were conserved in all these proteins. Thus, P. aeruginosa aminopeptidase may belong to the same family (M28) of metalloproteases.

Amino Acid Sequence↗
PubMed2003

Human leukocyte-derived arginine aminopeptidase. The third member of the oxytocinase subfamily of aminopeptidases.

In this study we report the cloning and characterization of a novel human aminopeptidase, which we designate leukocyte-derived arginine aminopeptidase (L-RAP). The sequence encodes a 960-amino acid protein with significant homology to placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase. The predicted L-RAP contains the HEXXH(X)18E zinc-binding motif, which is characteristic of the M1 family of zinc metallopeptidases. Phylogenetic analysis indicates that L-RAP forms a distinct subfamily with placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in the M1 family. Immunocytochemical analysis indicates that L-RAP is located in the lumenal side of the endoplasmic reticulum. Among various synthetic substrates tested, L-RAP revealed a preference for arginine, establishing that the enzyme is a novel arginine aminopeptidase with restricted substrate specificity. In addition to natural hormones such as angiotensin III and kallidin, L-RAP cleaved various N-terminal extended precursors to major histocompatibility complex class I-presented antigenic peptides. Like other proteins involved in antigen presentation, L-RAP is induced by interferon-gamma. These results indicate that L-RAP is a novel aminopeptidase that can trim the N-terminal extended precursors to antigenic peptides in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed2005

Identification of human aminopeptidase O, a novel metalloprotease with structural similarity to aminopeptidase B and leukotriene A4 hydrolase.

We have cloned and characterized a human brain cDNA encoding a new metalloprotease that has been called aminopeptidase O (AP-O). AP-O exhibits a series of structural features characteristic of aminopeptidases, including a conserved catalytic domain with a zinc-binding site (HEXXHX18E) that allows its classification in the M1 family of metallopeptidases or gluzincins. The structural complexity of AP-O is further increased by the presence of an additional C-terminal domain 170 residues long, which is predicted to have an ARM repeat fold originally identified in the Drosophila segment polarity gene product Armadillo. This ARM repeat domain is also present in aminopeptidase B, aminopeptidase B-like, and leukotriene A4 hydrolase and defines a novel subfamily of aminopeptidases that we have called ARM aminopeptidases. Northern blot analysis revealed that AP-O is mainly expressed in the pancreas, placenta, liver, testis, and heart. Human AP-O was produced in Escherichia coli, and the purified recombinant protein hydrolyzed synthetic substrates used for assaying aminopeptidase activity. This activity was abolished by general inhibitors of metalloproteases and specific inhibitors of aminopeptidases. Recombinant AP-O also cleaved angiotensin III to generate angiotensin IV, a bioactive peptide of the renin-angiotensin pathway with multiple actions on diverse tissues, including brain, testis, and heart. On the basis of these results we suggest that AP-O could play a role in the proteolytic processing of bioactive peptides in those tissues where it is expressed.

Amino Acid Sequence↗
PubMed1995

Degradation of dynorphin-related peptides by the puromycin-sensitive aminopeptidase and aminopeptidase M.

The degradation of dynorphin-related peptides by the puromycin-sensitive aminopeptidase and aminopeptidase M was examined using these peptides as alternate substrate inhibitors. Ki determinations showed that both aminopeptidases exhibit a higher affinity for longer dynorphin-related peptides, i.e., Ki for dynorphin A-17 = 23-30 nM with the Ki increasing to 25-50 microM for the enkephalin pentapeptides. Binding appears dependent not only on peptide length, but also on its sequence. With aminopeptidase M, as the peptide size increases from five to 10 amino acids, kcat remains relatively constant; however, as the peptide size increases beyond a decapeptide, kcat decreases significantly. With the puromycin-sensitive aminopeptidase, similar results were obtained except that kcat was greatest for the pentapeptide. Thus, if one considers kcat/Km as the relevant kinetic constant for estimating in vivo peptide hydrolysis, these results are consistent with the involvement of aminopeptidase M and the puromycin-sensitive aminopeptidase in the degradation of extended dynorphin-related peptides.

Amino Acid Sequence↗