SEARCH · PubMed Health

Results for “AMINOPEPTIDASES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37Linked to original sources

PubMedDate not supplied

Demonstration of placental aminopeptidase isozymes by disc electrophoresis and its application to the forensic pregnancy test from blood stains.

Serum and placental aminopeptidase isozymes were characterized by means of disc electrophoresis. Human pregnancy sera exhibited three bands with leucine aminopeptidase (LAP) activity. The fastest moving LAP band was also present in nonpregnancy and foetal sera. The other two cystine aminopeptidase bands (CAP1 and CAP2), characteristic of pregnancy, were demonstrated in lysosomal extracts and hence suggested to be derived from the lysosomes of the placenta. These two CAP bands of lysosomal origin appeared in sera of pregnant women at different times during the course of gestation; CAP1 band in the 3rd month and CAP2 band in the 6th month. The information obtained from serum samples was applied to the medicolegal diagnosis of pregnancy from blood stains. Excess haemoglobin in blood stain extracts could be eliminated by subjecting them to a P-cellulose column. The results and their implications were discussed. This technique proved to be of practical use in the examination of blood stains up to periods of about 2 weeks after stain formation.

Aminopeptidases↗

PubMed2005

The oxytocinase subfamily of M1 aminopeptidases.

The placental leucine aminopeptidase (P-LAP), adipocyte-derived leucine aminopeptidase (A-LAP) and leukocyte-derived aminopeptidase (L-RAP) belong to one distinct group of the M1 family of amimopeptidases, which we term the "Oxytocinase subfamily". They share HEXXH(X)18E Zn-binding and GAMEN motifs essential for the enzymatic activities. Intracellular localization is the characteristic feature of the subfamily members. While P-LAP is translocated from intracellular vesicles to plasma membrane in a stimulus-dependent manner, both A-LAP and L-RAP are retained in the endoplasmic reticulum. They contain sequences necessary for the specific localization in the cell. It is getting evident that the subfamily members play important roles in the maintenance of homeostasis including maintenance of normal pregnancy, memory retention, blood pressure regulation and antigen presentation. In this review, current situation of this newly identified subfamily is summarized.

Amino Acid Sequence↗

PubMed2005

Proteolytic activity in Encephalitozoon cuniculi sporogonial stages: predominance of metallopeptidases including an aminopeptidase-P-like enzyme.

A fraction enriched in spore precursor cells (sporoblasts) of the microsporidian Encephalitozoon cuniculi, an intracellular parasite of mammals, was obtained by Percoll gradient centrifugation. Soluble extracts of these cells exhibited proteolytic activity towards azocasein, with an alkaline optimum pH range (9-10). Prevalence of some metallopeptidases was supported by the stimulating effect of Ca2+, Mg2+, Mn2+ and Zn2+ ions, and inhibition by two chelating agents (EDTA and 1,10-phenanthroline), a thiol reductant (dithiothreitol) and two aminopeptidase inhibitors (bestatin and apstatin). Zymographic analysis revealed four caseinolytic bands at about 76, 70, 55 and 50 kDa. Mass spectrometry of tryptic peptides from one-dimensional gel slices identified a cytosol (leucine) aminopeptidase homologue (M17 family) in 50-kDa band and an enzyme similar to aminopeptidase P (AP-P) of cytosolic type (M24B subfamily) in 70-kDa band. Multiple sequence alignments showed conservation of critical residues for catalysis and metal binding. A long insertion in a common position was found in AP-P sequences from E. cuniculi and Nosema locustae, an insect-infecting microsporidian. The expression of cytosolic AP-P in sporogonial stages of microsporidia may suggest a key role in the attack of proline-containing peptides as a prerequisite to long-duration biosynthesis of structural proteins destined to the sporal polar tube.

Amino Acid Sequence↗

PubMed2003

Homology modeling and calculation of the cobalt cluster charges of the Encephazlitozoon cuniculi methionine aminopeptidase, a potential target for drug design.

Fumagillin is a potent anti-angiogenic drug used in cancer treatments. It is also one of the few molecules active against the Enterocytozoon and Encephalitozoon parasites responsible for various clinical syndromes in HIV-infected or immunosuppressive treated patients. Its toxicity, however, makes desirable the design of more specific molecules. The fumagillin target, as anti-angiogenic agent, is the methionine aminopeptidase, an ubiquitous metallo-enzyme responsible for the removing of the N-terminal methionine in nascent proteins. By analogy, it has been proposed that this enzyme could also be the target in the parasites. As a first approach to verify this and to determine if it would be possible to design a more specific derivative, we have built a homology model of the E. cuniculi aminopeptidase. The charges of the two cobalt ions present in the active site and of the side-chains involved in their binding were computed using ab-initio methods. A preliminary comparison of the interactions of the fumagillin and of a related compound, the TNP-470, with both the human and the parasitic enzymes strongly support the hypothesis that the parasitic aminopeptidase is indeed the target of the fumagillin. It also suggests that the TNP-470 interact identically with both enzymes while there could be small differences in case of the fumagillin.

Amino Acid Sequence↗

PubMed2002

Ethanol modifies differently aspartyl- and glutamyl-aminopeptidase activities in mouse frontal cortex synaptosomes.

Aminopeptidase A activity (aspartyl aminopeptidase (AspAP) and glutamyl aminopeptidase (GluAP) exerts angiotensinase activity due to its relation to the metabolism of angiotensins in the regional brain renin-angiotensin system (RAS). This activity may also modify the free amino acid pool through the release of N-terminal acidic amino acids. Ethanol (EtOH) exerts profound effects on the brain, inducing important neurological damages. Our purpose is to study the influence of EtOH on AspAP and GluAP activities on basal and K(+)-stimulated conditions, at the synapse level. We used mouse frontal cortex synaptosomes and their incubation supernatant in a Ca(2+)-containing or Ca(2+)-free artificial cerebrospinal fluid. We evaluate the possible contribution of these enzymatic activities on brain blood pressure regulation through RAS and/or the free acidic amino acid pool. The results obtained are correlated with several parameters of oxidative stress, such as free radical generation, lipid peroxidation, and protein oxidation. Under basal conditions, in synaptosomes, EtOH inhibits AspAP and GluAP activities independently of Ca(2+). In the supernatant, however, EtOH differently modulates the two enzyme activities under the various concentrations. Under K(+)-stimulated conditions, EtOH inhibits the K(+)-stimulated increase on AspAP and GluAP differently depending on the presence or absence of Ca(2+) and the concentration of EtOH used. These results invalidate the idea that excess free acidic amino acids could be released by AspAP and GluAP to induce neurodegeneration. The changes in AspAP and GluAP activities as a consequence of EtOH administration and their role in the brain RAS are discussed.

Amino Acids↗

PubMed1989

Inhibition of aminopeptidases by aminophosphonates.

More than 30 aminophosphonates were synthesized to probe how the structural changes introduced into the phosphonic acid analogue of leucine, a potent inhibitor of cytosolic leucine aminopeptidase (Giannousis & Bartlett, 1987), affect their ability to inhibit cytosolic (EC 3.4.11.1) and microsomal (EC 3.4.11.2) aminopeptidases. Although most of the compounds studied were found to exert only a modest competitive inhibitory effect, nearly every modification of the structure of the phosphonic acid analogue of leucine was reflected in a marked difference in the affinities of these compounds for the two enzymes. [1-Amino-2-(N-alkylamino)ethyl]phosphonic acids are effective inhibitors of the microsomal enzyme, acting in a time-dependent manner. Kinetic data obtained for these inhibitors correspond to the mechanism for a biphasic slow-binding inhibition process: E + I in equilibrium E* in equilibrium E*I, in which the slow initial isomerization of the enzyme is followed by the fast formation of enzyme-inhibitor complex. The most effective inhibitor of this type was [1-amino-2-(N-cyclohexylamino)ethyl]phosphonic acid, which has a Ki value of 0.87 microM toward the microsomal aminopeptidase--a value that can be considered as equipotent with bestatin and with leucinal and hydroxamic acids, the strongest known nonpeptide inhibitors of this enzyme.

Aminopeptidases↗

PubMed1999

Insights into the mechanism of Escherichia coli methionine aminopeptidase from the structural analysis of reaction products and phosphorus-based transition-state analogues.

In an effort to differentiate between alternative mechanistic schemes that have been postulated for Escherichia coli methionine aminopeptidase (eMetAP), the modes of binding of a series of products and phosphorus-based transition-state analogues were determined by X-ray crystallography. Methionine phosphonate, norleucine phosphonate, and methionine phosphinate bind with the N-terminal group interacting with Co2 and with the respective phosphorus oxygens binding between the metals, interacting in a bifurcated manner with Co1 and His178 and hydrogen bonded to His79. In contrast, the reaction product methionine and its analogue trifluoromethionine lose interactions with Co1 and His79. The interactions with the transition-state analogues are, in general, very similar to those seen previously for the complex of the enzyme with a bestatin-based inhibitor. The mode of interaction of His79 is, however, different. In the case of the bestatin-based inhibitor, His79 interacts with atoms in the peptide bond between the P(1)' and P(2)' residues. In the present transition-state analogues, however, the histidine moves 1.2 A toward the metal center and hydrogen bonds with the atom that corresponds to the nitrogen of the scissile peptide bond (i.e., between the P(1) and P(1)' residues). These observations tend to support one of the mechanistic schemes for eMetAP considered before, although with a revision in the role played by His79. The results also suggest parallels between the mechanism of action of methionine aminopeptidase and other "pita-bread" enzymes including aminopeptidase P and creatinase.

Amino Acid Substitution↗

PubMed1984

Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes.

Amastatin [(2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl-L-valyl-L-valyl-L- aspartic acid] and bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine] are slow-binding, competitive inhibitors of aminopeptidase M (AP-M) with net inhibition constants (Ki) of 1.9 X 10(-8) and 4.1 X 10(-6) M, respectively. The effect of inhibitor structure on net Ki and on slow-binding inhibition was evaluated for analogues of both inhibitors on AP-M and leucine aminopeptidase (LAP). The (2S)-hydroxyl group contributes to the stabilization of a collision complex [EI], which is formed rapidly. In contrast, increasing the peptide chain length of the inhibitor produces more potent inhibitors as a consequence of a slower binding process. A statine analogue of amastatin [(3S,4S)-Sta-Val-Val-Asp] stimulated rather than inhibited LAP. AP-M binds tri- and tetrapeptide inhibitors more strongly than dipeptide inhibitors, whereas LAP binds dipeptide inhibitors more strongly. The difference in binding can be used to distinguish cytosolic from membrane-bound aminopeptidases.

Aminopeptidases↗

PubMed2002

Investigation of the metal binding site in methionine aminopeptidase by density functional theory.

All methionine aminopeptidases exhibit the same conserved metal binding site. The structure of this site with either Co2+ ions or Zn2+ ions was investigated using density functional theory. The calculations showed that the structure of the site was not influenced by the identity of the metal ions. This was the case for both of the systems studied; one based on the X-ray structure of the human methionine aminopeptidase type 2 (hMetAP-2) and the other based on the X-ray structure of the E. coli methionine aminopeptidase type I (eMetAP- 1). Another important structural issue is the identity of the bridging oxygen, which is part of either a water molecule or a hydroxide ion. Within the site of hMetAP-2 the results strongly indicate that a hydroxide ion bridges the metal ions. By contrast, the nature of the oxygen bridging the metal ions within the metal binding site of eMetAP-1 cannot be determined based on the results here, due to the similar structural results obtained with a bridging water molecule and a bridging hydroxide ion.

Aminopeptidases↗

PubMedDate not supplied

Binding of alpha-hydroxy-beta-amino acid inhibitors to methionine aminopeptidase. The performance of two types of scoring functions.

The binding mode of a recently described set of alpha-hydroxy-beta-amino acid inhibitors of methionine aminopeptidase type 2 is suggested in the present work. The binding mode is supported by analysis of published structures of transition state analogues co-crystallised with E. coli methionine aminopeptidase and by a comparison of molecular interaction fields calculated using GRID for E. coli and human methionine aminopeptidase. Based on the suggested binding mode two types of scoring functions have been evaluated and compared. These are the commercially available consensus score, CScore, and scoring of the ligands employing energies calculated using the Merck Molecular Force Field (MMFF). Enriched subsets of ligands were obtained when using CScore, but these scores could not be used to assess the relative affinities of individual compounds. Although still not sufficiently accurate for reliable predictive purposes, an improved correlation was obtained between structure and affinity using a combined force field energy including contributions from solvation and conformational energy penalty for binding.

Algorithms↗

PubMed2000

Structure of the human oxytocinase/insulin-regulated aminopeptidase gene and localization to chromosome 5q21.

The human oxytocinase/insulin-regulated aminopeptidase (OTase/IRAP) is a 1024 amino acid type II integral membrane protein that is expressed mainly in fat, muscle and placenta tissues. It has been thought to be involved mainly in the control of onset of labour but recently rat OTase/IRAP was shown to participate in the regulation of glucose transporter isoform 4 vesicle trafficking in adipocytes as well. To approach an understanding of OTase/IRAP gene regulation the organization of the human gene was determined. Accordingly, three overlapping genomic clones were isolated and characterized. The human OTase/IRAP gene (OTASE) was found to span approximately 75 kb containing 18 exons and 17 introns. The gluzincin aminopeptidase motif: GAMEN-(31 amino acids)-HELAH-(18 amino acids)-E associated with Zn2+-binding, substrate binding and catalysis is encoded by exons 6 and 7. A major and a minor transcriptional initiation site in OTASE were identified by primer extension 514 bp and 551 bp, respectively, upstream of the translation start codon. Chloroamphenicol acetyltransferase-reporter assays revealed a functional CpG-rich promoter/enhancer region located between nucleotide -621 and the major transcriptional initiation site. Human OTASE was assigned to chromosome 5 by hybridization to genomic DNA from characterized somatic cell hybrids. Finally, the OTASE and the human aminopeptidase A gene were subchromosomally localized to 5q21 and 4q25, respectively, by in situ hybridization.

Amino Acid Sequence↗

PubMed2001

Mutational analysis of the active site of human insulin-regulated aminopeptidase.

Insulin-regulated aminopeptidase (IRAP) is a type II integral membrane protein belonging to the gluzincin family of metallopeptidases identified by the characteristic Zn(2+)-coordination sequence element, HEXXH-(18-64X)-E. A second conserved sequence element, the GXMEN motif, positioned 22-32 amino acids N-terminal to the Zn(2+)-coordination sequence element distinguishes the gluzincin aminopeptidases from other gluzincins. To investigate the importance of the G428AMEN and H464ELAH-(18X)-E487 motifs for the activity of IRAP, mutational analysis was carried out. cDNA encoding the full-length transmembrane form of human IRAP was expressed in HEK293 cells and recombinant wild-type IRAP was shown to have biochemical and enzymatic properties similar to those reported for native IRAP and the soluble serum form of IRAP. Mutational analysis using single amino-acid substitutions in the GAMEN motif (G428A, A429G, M430K, M430E, M430I, E431D and E431A) and in the Zn(2+)-binding motif (H464Y, E465D, E465Q, H468Y, E487D and E487Q) resulted in decreased or abolished aminopeptidase activity towards the leucine-para-nitroanilide substrate. The results show that conservation of residues within the GAMEN and Zn(2+)-binding motifs is important for IRAP enzyme activity.

Amino Acid Motifs↗

PubMed2000

Placental aminopeptidase A as a possible barrier of angiotensin II between mother and fetus.

Aminopeptidase A (AP-A EC 3.4.11.7), which is a membrane-bound zinc metalloprotease, is present in the placenta. AP-A selectively hydrolyzes N-terminal glutamyl and aspartyl residues and cleaves angiotensin II to form angiotensin III. To determine the role of placental aminopeptidase A under physiological and pathological conditions, we evaluated its immunolocalization and enzymatic activities in the placenta. AP-A was localized in the basal zone of the syncytiotrophoblast, in the membranes of the cytotrophoblast, and in fetal arterioles and venules within the stem villi. AP-A activity in the microsomal fraction of placental villi seemed to be remained essentially constant throughout gestation. The renin-angiotensin system is considered to be accelerated in pre-eclampsia. This AP-A activity was higher in pre-eclampsia (2.86+/-0.30 nmol beta NA/mg protein/h) than in uncomplicated pregnancy from 28 to 41 weeks of gestation (2.08+/-0.18 nmol beta NA/mg protein/h). Angiotensin II evoked AP-A activity in first trimester trophoblast, and Losartan and PD 123177 in combination significantly inhibited this induction of AP-A activity. The results of immunohistochemical evaluation and enzymatic activity suggested that placental aminopeptidase A may play a role as a component of the barrier of angiotensin II between mother and fetus.

Adult↗

PubMed1992

Induction by cortisol of aminopeptidases production from the human placenta in tissue culture.

Human placental aminopeptidase M and A and post-proline endopeptidase are known to act as degrading enzymes of bioactive peptides such as angiotensin II, oxytocin and endogenous opioids. We tested the effects of cortisol on the activities of human placental aminopeptidase A and M and post-proline endopeptidase using short-cultured placental tissues. From 34.5 nM to 3.45 microM of cortisol significantly increased the activities of 3 enzymes. Our present data suggest a possible important role of cortisol in the growth of human placenta via induction of placental aminopeptidases.

Aminopeptidases↗

PubMed1995

Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases.

We previously characterized a methionine aminopeptidase (EC 3.4.11.18; Met-AP1; also called peptidase M) in Saccharomyces cerevisiae, which differs from its prokaryotic homologues in that it (i) contains an N-terminal zinc-finger domain and (ii) does not produce lethality when disrupted, although it does slow growth dramatically; it is encoded by a gene called MAP1. Here we describe a second methionine aminopeptidase (Met-AP2) in S. cerevisiae, encoded by MAP2, which was cloned as a suppressor of the slow-growth phenotype of the map1 null strain. The DNA sequence of MAP2 encodes a protein of 421 amino acids that shows 22% identity with the sequence of yeast Met-AP1. Surprisingly, comparison with sequences in the GenBank data base showed that the product of MAP2 has even greater homology (55% identity) with rat p67, which was characterized as an initiation factor 2-associated protein but not yet shown to have Met-AP activity. Transformants of map1 null cells expressing MAP2 in a high-copy-number plasmid contained 3- to 12-fold increases in Met-AP activity on different peptide substrates. The epitope-tagged suppressor gene product was purified by immunoaffinity chromatography and shown to contain Met-AP activity. To evaluate the physiological significance of Met-AP2, the MAP2 gene was deleted from wild-type and map1 null yeast strains. The map2 null strain, like the map1 null strain, is viable but with a slower growth rate. The map1, map2 double-null strains are nonviable. Thus, removal of N-terminal methionine is an essential function in yeast, as in prokaryotes, but yeast require two methionine aminopeptidases to provide the essential function which can only be partially provided by Met-AP1 or Met-AP2 alone.

Amino Acid Sequence↗

PubMed1996

Characterization of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes.

A novel membrane aminopeptidase has been identified as a major protein in vesicles from rat adipocytes containing the glucose transporter isotype Glut4. In this study we have characterized this aminopeptidase, referred to as vp165, in 3T3-L1 adipocytes. The subcellular distributions of vp165 and Glut4 were determined by immunoisolation of vesicles with antibodies against both proteins, by immunofluorescence, and by subcellular fractionation and immunoblotting. Relative amounts of vp165 at the cell surface in basal and insulin-treated cells were assayed by cell surface biotinylation. These experiments showed that vp165 and Glut4 were entirely colocalized and that vp165 increased markedly at the cell surface in response to insulin, in a way similar to Glut4. When intact cells were assayed with a novel, membrane-impermeant fluorogenic substrate for vp165, we found that insulin stimulated aminopeptidase activity at the cell surface. This observation provides direct evidence for the functional consequence of vp165 translocation.

3T3 Cells↗

PubMed2002

Mice deficient in the insulin-regulated membrane aminopeptidase show substantial decreases in glucose transporter GLUT4 levels but maintain normal glucose homeostasis.

The insulin-regulated aminopeptidase (IRAP) is a zinc-dependent membrane aminopeptidase. It is the homologue of the human placental leucine aminopeptidase. In fat and muscle cells, IRAP colocalizes with the insulin-responsive glucose transporter GLUT4 in intracellular vesicles and redistributes to the cell surface in response to insulin, as GLUT4 does. To address the question of the physiological function of IRAP, we generated mice with a targeted disruption of the IRAP gene (IRAP-/-). Herein, we describe the characterization of these mice with regard to glucose homeostasis and regulation of GLUT4. Fed and fasted blood glucose and insulin levels in the IRAP-/- mice were normal. Whereas IRAP-/- mice responded to glucose administration like control mice, they exhibited an impaired response to insulin. Basal and insulin-stimulated glucose uptake in extensor digitorum longus muscle, and adipocytes isolated from IRAP-/- mice were decreased by 30-60% but were normal for soleus muscle from male IRAP-/- mice. Total GLUT4 levels were diminished by 40-85% in the IRAP-/- mice in the different muscles and in adipocytes. The relative distribution of GLUT4 in subcellular fractions of basal and insulin-stimulated IRAP-/- adipocytes was the same as in control cells. We conclude that IRAP-/- mice maintain normal glucose homeostasis despite decreased glucose uptake into muscle and fat cells. The absence of IRAP does not affect the subcellular distribution of GLUT4 in adipocytes. However, it leads to substantial decreases in GLUT4 expression.

Adipocytes↗

PubMed1990

Aminopeptidase-induced elevations and reductions in blood pressure in the spontaneously hypertensive rat.

In vitro results indicated that human placenta-derived aminopeptidase A (APA) was very effective at hydrolyzing aspartate from the angiotensin molecule, thus converting angiotensin II to angiotensin III, but was not active against angiotensin III. In vivo experiments revealed significant elevations in blood pressure when APA was intracerebroventricularly infused into anesthetized spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive control rats (WKY), with maximum mean (+/- s.e.m.) increases of 30.0 +/- 2.5 and 32.5 +/- 3.7 mmHg, respectively. By contrast, in vitro incubation results utilizing leucine aminopeptidase M (LAP-M) indicated very active degradation of angiotensin III, with less rapid degradation of angiotensin II. The intracerebroventricular infusion of LAP-M significantly reduced blood pressure, particularly in the SHR, but also in WKY, -65.8 +/- 5.1 and -42.5 +/- 6.1 mmHg, respectively. Pretreatment with the specific angiotensin receptor antagonist, Sar1, Thr8 angiotensin II (sarthran) significantly diminished the subsequent APA-induced increase in blood pressure in members of both strains. Pretreatment with sarthran has previously been shown to significantly diminish LAP-M-induced decreases in blood pressure in SHR. Thus, the effects of these aminopeptidases appear to be primarily dependent upon the brain angiotensinergic system, and are consistent with the hypothesis that angiotensin III is the primary active form of central angiotensin.

Aminopeptidases↗