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PubMed1993

Two cell-wall-associated aminopeptidases from Lactobacillus helveticus and the purification and characterization of APII from strain ITGL1.

Lactobacillus helveticus ITGL1 is able to hydrolyse many amino-acyl and dipeptidyl-p-nitroanilides. Analysis of heat inactivation kinetics, metal ion and protease inhibitor effects, and the subcellular location of aminopeptidase activities in both the parental strain and mutants deficient in lysyl-p-nitroanilide hydrolysis, led to the characterization of two cell-wall-associated aminopeptidases, APII and APIV. APII, which catalysed L-lysine p-nitroanilide hydrolysis, was purified about 28-fold to homogeneity from cell-wall extracts of L. helveticus ITGL1 and characterized. The purified enzyme appeared to be monomeric, with a molecular mass of 97 kDa. Aminopeptidase activity was greatest at pH 6.5 and 50 degrees C. APII was completely inhibited by bestatin, chelating agents such as EDTA or 1,10-phenanthroline and the divalent cations Zn2+ and Cu2+. The activity of the EDTA-treated enzyme was restored by Co2+, Ca2+ or Mn2+. Although APII was able to degrade several dipeptides and tripeptides with hydrophobic N-terminal amino acid (Leu, Ala), it was inactive on peptides containing Pro or Gly, and may thus contribute to the development of cheese flavour by processing bitter peptides.

Amino Acid Sequence↗

PubMed1984

Aminopeptidase A in reproductive organs of the male rat: evidence for high activity in the posterior lobe of the prostate.

Of the reproductive organs of the male rat a high level of hydrolysis of alpha-L-glutamyl-beta-napthylamide (GluNA) and alpha-L-aspartyl-beta-naphthylamide (AspNA) in the presence of Ca2+ (5mM) (aminopeptidase A) was found in the posterior lobe of the prostate. Histochemically, this enzyme was localized in the epithelial cells of acini, which were grouped in the dorsal part and sharply separated from non-active acini in the anterior part of the lobe. A single peak of Ca2+-activated GluNA and AspNA hydrolysis was obtained after chromatofocusing at pI 4.9 and on anion exchange chromatography this activity eluted at 0.09 M NaCl. After gel filtration on Sepharose 6B a major peak of activity was found at the elution volume (Ve/Vo = 2.28). In all of these fractionation procedures aminopeptidase A was partially or totally overlapped by other aminopeptidases hydrolysing various amino acid-beta-naphthylamides. A pooled enzyme preparation gave an optimum at pH 7.3. The hydrolysis of GluNA was markedly enhanced in the presence of Ba2+, Ca2+ and Sr2+, but the hydrolysis of AspNA was activated only by Ca2+ and Sr2+. Castration caused a significant decrease in the hydrolysis of GluNA by the posterior lobe, but did not influence the low levels of activity in other parts of the prostate or in the seminal vesicles.

Amino Acids↗

PubMed2005

Characterization of two new aminopeptidases in Escherichia coli.

Two genes in the Escherichia coli genome, ypdE and ypdF, have been cloned and expressed, and their products have been purified. YpdF is shown to be a metalloenzyme with Xaa-Pro aminopeptidase activity and limited methionine aminopeptidase activity. Genes homologous to ypdF are widely distributed in bacterial species. The unique feature in the sequences of the products of these genes is a conserved C-terminal domain and a variable N-terminal domain. Full or partial deletion of the N terminus in YpdF leads to the loss of enzymatic activity. The conserved C-terminal domain is homologous to that of the methionyl aminopeptidase (encoded by map) in E. coli. However, YpdF and Map differ in their preference for the amino acid next to the initial methionine in the peptide substrates. The implication of this difference is discussed. ypdE is the immediate downstream gene of ypdF, and its start codon overlaps with the stop codon of ypdF by 1 base. YpdE is shown to be a metalloaminopeptidase and has a broad exoaminopeptidase activity.

Amino Acid Sequence↗

PubMed1999

PC18, a specific aminopeptidase N inhibitor, induces vasopressin release by increasing the half-life of brain angiotensin III.

Angiotensin III (AngIII), which is metabolized in vivo by aminopeptidase N (APN), was previously shown to be one of the main effector peptides of the brain renin-angiotensin system (RAS) in the control of vasopressin release. Recently, a potent APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol, methionine thiol), has been developed. In this study, we first checked the in vitro selectivity of PC18 towards APN, aminopeptidase A (APA) and aminopeptidase B (APB), three zinc metalloproteases with significant identity between their amino acid sequences. The Ki values of this compound on APN were found to be in the nanomolar range (Ki = 8.0 +/- 1.7 nM) but it was 2,150 and 125 times less active on APA and APB, respectively. Secondly, we evaluated in vivo the effect of brain APN inhibition with PC18 on the inactivation of brain AngIII and on vasopressin secretion in mice. For this purpose, mice received [3H]AngII intracerebroventricularly in the presence or absence of the APN inhibitor PC18 (30 microg). At different times after the injection, [3H]AngIII levels were evaluated from hypothalamus homogenates after separation by cation-exchange chromatography. PC18 induced an accumulation of [3H]AngIII, increasing its half-life 3.9 times as compared with control values. In addition, the effect of PC18 on vasopressin release was studied in mice. PC18 (10-100 microgram) was injected intracerebroventricularly, and plasma vasopressin levels were estimated by radioimmunoassay. PC18 increased vasopressin levels in a dose-dependent manner. The maximal increase in vasopressin release (+220%) is observed for a dose of PC18 of 100 microgram and was inhibited 75% by the coadministration of the AngII receptor antagonist (Sar1-Ala8)-AngII (0.5 microgram). These results indicate that in vivo, in the mouse brain, APN inhibition by PC18 increases the half-life of endogenous AngIII, resulting in an enhanced vasopressin release.

Aminopeptidases↗

PubMed1992

Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae.

A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced. The DNA sequence encodes a precursor protein containing 387 amino acid residues. The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids. The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established. In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases. Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells.

Amino Acid Sequence↗

PubMed1990

Role of aminopeptidase activity in the regulation of the pressor activity of circulating angiotensins.

Aminopeptidase A (AmA; EC 3.4.11.7) can convert angiotensin II (AII) to angiotensin III (AIII), and aminopeptidase M (AmM; EC 3.4.11.2) has been shown to degrade AIII. The present study investigated angiotensin metabolism by AmA and AmM activities in rat plasma. Plasma AmA and AmM activities hydrolyzed glutamyl-2-naphthylamide and alanyl-2-naphthylamide at rates of 10.6 and 30.0 nmol/min/ml, respectively. Plasma hydrolysis of AII (4.1 +/- 0.5 nmol/min/ml) was only one-third as rapid as AIII (13.3 +/- 1.7 nmol/min/ml). The Km of AII and AIII for AmA and AmM were 90.3 +/- 14.3 and 29.5 +/- 8.2 microM, respectively. The aminopeptidase inhibitor amastatin was 40-fold more potent as an inhibitor of AmM activity (IC50 = 0.2 microM) than of AmA activity (IC50 = 8 microM). In order to examine metabolism in vivo, blood pressure responses to angiotensins were obtained in anesthesized rats before and during infusion of amastatin (16 nmol/min i.v.). Amastatin specifically inhibited plasma AmM and AmA activities 81 and 10%, respectively. Consistent with the lower inhibition of AmA, the potency of angiotensin I and AII were only slightly increased after amastatin. However, the potency of AIII and des(Asp1)angiotensin I were significantly increased regarding both maximal change in blood pressure and duration of action. These data support an important role for both AmA and AmM activities in the metabolism of circulating angiotensins and establish both the value and limitations of amastatin as an inhibitor of peripheral angiotensin metabolism.

Aminopeptidases↗

PubMedDate not supplied

[Cerebral aminopeptidase activity: effect of the administration of imipramine].

Imipramine is widely accepted as an effective antidepressant treatment. In this paper, changes in Lys- and Leu-aminopeptidase activities in several rat brain regions after imipramine treatment are described. Decreases of Lys-aminopeptidase activity in the olphactory bulbus, cerebellum, hypothalamus and pituitary gland were observed. In the same way, there were a decreases in Leu-aminopeptidase activity in the parietal and occipital cortices, cerebellum thalamus, hypothalamus and pituitary gland. It is suggested that these enzyme activities play a part in the imipramine action mechanism, possibly by regulating the activity of several neuroactive peptides.

Aminopeptidases↗

PubMed1989

Pre- and postnatal aminopeptidase activities in the rat brain.

Research concerning the functional role of brain peptides is performed, in part, by studying peptidase enzymes which might be involved in brain peptide processing or inactivation. Aminopeptidase (AP) activity has been proposed as a candidate regulator of the degradation of these peptides. In this paper, changes in Lys- and Leu-aminopeptidase activities in rat brain hemispheres, cerebellum and medulla were examined in 20 day fetuses and one day postnatal subjects. Aminopeptidase activities were studied by measuring the rate of hydrolysis of the artificial substrates Lys- and Leu-2-naphthylamides (fluorimetrically detected in triplicate). Both enzyme activities increase from the last fetal stage up to the first day of birth in all the brain areas examined except for the case of Leu-AP activity in the medulla. It is suggested that these activities play a part in the neurochemical changes that take place during rat brain maturation, possibly by regulating the activity of several neuroactive peptides.

Aminopeptidases↗

PubMed1985

Amino-terminal processing of mutant forms of yeast iso-1-cytochrome c. The specificities of methionine aminopeptidase and acetyltransferase.

Amino-terminal processing in the yeast Saccharomyces cerevisiae has been investigated by examining numerous mutationally altered forms of iso-1-cytochrome c. Amino-terminal residues of methionine were retained in sequences having penultimate residues of arginine, asparagine, glutamine, isoleucine, leucine, lysine, and methionine; in contrast, the amino-terminal methionine residues were exercised from residues of alanine, glycine, and threonine and were partially excised from residues of valine. The results suggest the occurrence of a yeast aminopeptidase that removes amino-terminal residues of methionine when they precede certain amino acids. A systematic search of the literature for amino-terminal sequences formed at initiation sites suggests the hypothetical yeast aminopeptidase usually has the same specificity as the amino peptidase from bacteria and higher eukaryotes. Our results and the results from the literature search suggest that the aminopeptidase cleaves amino-terminal methionine when it precedes residues of alanine, glycine, proline, serine, threonine, and valine but not when it precedes residues of arginine, asparagine, aspartic acid, glutamine glutamic acid, isoleucine, leucine, lysine, or methionine. In contrast to the normal iso-1-cytochrome c and in contrast to the majority of the mutationally altered proteins, certain forms were acetylated including the following sequences: acetyl(Ac)-Met-Ile-Arg-, Ac-Met-Ile-Lys, Ac-Met-Met-Asn-, and Ac-Met-Asn-Asn-. We suggest yeast contains acetyltransferases that acetylates these mutant forms of iso-1-cytochromes c because their amino-terminal regions resemble the amino-terminal regions of natural occurring proteins which are normally acetylated. The lack of acetylation of closely related sequences suggest that the hypothetical acetyltransferases are specific for certain amino-terminal sequences and that the 3 amino-terminal residues may play a critical role in determining these specificities.

Acetyltransferases↗

PubMed1986

The dissociation between the plasma levels of aminopeptidases A and B in spontaneously hypertensive rats.

We performed a longitudinal study for 20 weeks on spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKR) to determine the relationship between peptide metabolism and the age-dependent increase in blood pressure. In both SHR and WKR, the plasma level of aminopeptidase A (AP-A) clearly showed an age-dependent decrease. The plasma level of aminopeptidase B paralleled that of AP-A in WKR, but such an age-dependency was not observed in SHR, thus showing a dissociation between the two aminopeptidases. With age in both strains, the level of angiotensin-converting enzyme tended to decrease, while that of kallikrein activity tended to increase. In addition to these findings, a multivariate study testing the relationship of blood pressure to these enzyme activities, as well as to plasma levels of angiotensin I and renin activity, suggested abnormalities in the networks of proteolytic enzymes and in the peptide metabolism surrounding the renin-angiotensin system in SHR. These abnormalities may play some important roles in pathophysiological mechanisms of hypertension in SHR.

Aging↗

PubMed1978

Isolation of aminopeptidases from Histoplasma capsulatum.

Two aminopeptidases (arylamidases) were isolated and partially purified from Histoplasma capsulatum. The larger molecular weight enzyme was a proline iminopeptidase and hydrolyzed primarily a synthetic substrate, L-prolyl-beta-napthylamide. The other aminopeptidase was less substrate specific and hydrolyzed rapidly the following amino acid beta-napthylamides (beta NA): L-arginyl-beta NA greater than L-lysyl-beta NA greater than -L-4-methoxy-leucyl-beta NA greater than L-leucyl-beta NA greater than L-phenylalanyl-beta NA greater than L-alanyl-beta NA. The proline iminopeptidase was purified 1420 fold while the leucine aminopeptidase was purified 650 fold with good recovery.

Aminopeptidases↗

PubMed1976

[Studies on the specificity of alanine aminopeptidase and its variants in the serum in hepatobiliary diseases].

After examinations on a mixed number of patients (236 patients) the increases of the activities of the alanine aminopeptidase in the serum with relatively high certainty may be traced back to diseases of the liver and of the bile ducts. The hepatobiliary specifity is disturbed only by generalised dermatological diseases, especially eczemas. Changes in the electrophoretic pattern of the serum alanine aminopeptidase are also relatively specific for diseases or lesions of the hepatobiliary system. In 11 of the 12 anomalous zymograms of the alanine aminopeptidase observed among 236 patients the aimed search resulted in an alteration of the hepatobiliary system.

Alkaline Phosphatase↗

PubMed1987

Leucine aminopeptidase in intracytoplasmic membranes of Acinetobacter calcoaceticus.

Cells of Acinetobacter calcoaceticus strain 69-V contain an aminopeptidase that cleaves L-leucine amide, leucylglycine or leucine hydrazide with high efficiency. Leucine 4-nitroanilide and hydrazide are hydrolyzed to less than 0.1% and 1%, resp. of leucine amide. Grown on acetate-NH4+ medium the activity of the enzyme in the cytoplasm is increased 5-fold compared with cells grown on a casamino acid medium or on yeast extract. In these cases the specific activity of the unpurified enzyme is about 5 nkat/mg for the cytoplasmic and membrane-bound enzyme species as well. Up to 30% of the aminopeptidase activity were found mainly in intracytoplasmic membranes, less in cytoplasmic membranes and only traces in outer membranes, presumably as contaminations. It is solubilized by detergents but not by high salt concentrations. An addition of antipain or Z-Ala2-Phe-CH3 before cell rupture did not change the distribution of the enzyme. A mixture of EDTA and 1.10-phenanthroline diminished the membrane-bound enzyme from 11.4% to 4.3% and leupeptin or E-64 increased it to 20%. The enzyme is regarded as leucine aminopeptidase (LAP) bound to intracytoplasmic membranes.

Acinetobacter↗

PubMed1990

Recovery of rat nasal mucosa from the effects of aminopeptidase inhibitors.

Aminopeptidase inhibitors may be useful for improving the systemic bioavailability of peptide drugs administered nasally or by other routes. Preferably, their effects would be rapidly reversible. The recovery of peptide hydrolytic activity after exposure of the rat nasal cavity to various aminopeptidase inhibitors was evaluated. Leucine enkephalin (0.1 mM) was used as a model peptide which is predominantly metabolized by aminopeptidases nasally. All experiments involved in situ perfusion of the rat nasal cavity with leucine enkephalin and the inhibitor for 90 min, followed by a washout with saline, and finally a second experimental phase of perfusion with leucine enkephalin but no inhibitor. Boroleucine (0.1 microM) was a potent inhibitor of leucine enkephalin metabolism, and, after its removal, the leucine enkephalin metabolism rate returned to control levels. Much higher concentrations of bestatin (0.1 mM) and puromycin (1 mM) did not inhibit leucine enkephalin metabolism as much as did boroleucine. Furthermore, when these inhibitors were washed out, the rates of leucine enkephalin disappearance rebounded to higher than control levels. With bestatin this could have been partially due to membrane disruption, as evidenced by significantly increased protein concentrations in the perfusates. However, protein release was much lower than that caused by sodium glycocholate, a nasal membrane permeation enhancer. In considering the use of peptidase inhibitors as pharmaceutical adjuvants for peptide drugs, the aminoboronic acid derivatives, including boroleucine, have the advantages of efficacy at very low concentrations and reversibility of effects.

Aminopeptidases↗

PubMed1991

Isolation and characterization of Schizosaccharomyces pombe mutants lacking aminopeptidase activity.

A mutant strain of the fission yeast Schizosaccharomyces pombe defective in aminopeptidase I was isolated by screening for lack of activity against the chromogenic substrate lysine-beta-naphthylamide in isolated colonies. 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 chromosomal gene mutation. Gene dosage experiments indicated that the mutation might reside in the structural gene of aminopeptidase I. No vital consequences of aminopeptidase I deficiency on cell life and sporulation could be detected. However, the enzyme seems to be involved in protein degradation under conditions of nutrient deprivation.

Alleles↗

PubMed1999

Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases.

We have developed a specific and efficient method for complete removal of polyhistidine purification tags (HisTags) from the N-termini of target proteins. The method is based on the use of the aminopeptidase dipeptidyl peptidase I (DPPI), either alone or in combination with glutamine cyclotransferase (GCT) and pyroglutamyl aminopeptidase (PGAP). In both cases, the HisTag is cleaved off by DPPI, which catalyzes a stepwise excision of a wide range of dipeptides from the N-terminus of a peptide chain. Some sequences, however, are resistant to DPPI cleavage and a number of mature proteins have nonsubstrate N-termini which protects them against digestion. For such proteins, HisTags composed of an even number of residues can be cleaved off by treatment with DPPI alone. When the target protein is unprotected against DPPI, a blocking group is generated enzymatically from a glutamine residue inserted between the HisTag and the target protein. A protein with a HisTag-Gln extension is incubated with both DPPI and GCT. As above, the polyhistidine sequence is cleaved off by DPPI, but when the glutamine residue appears in the N-terminus, it is immediately converted into a pyroglutamyl residue by an excess of GCT and further DPPI digestion is prevented. The desired sequence is finally obtained by excision of the pyroglutamyl residue with PGAP. All the enzymes employed can bind to immobilized metal affinity chromatography (IMAC) matrices, and in this paper we demonstrate a simple and highly effective process combining IMAC purification of His-tagged proteins, our aminopeptidase-based method for specific excision of HisTags and use of subtractive IMAC for removing processing enzymes. Typical recoveries were 75-90% for the enzymatic processing and subtractive IMAC. The integrated process holds promises for use in large-scale production of pharmaceutical proteins because of a simple overall design, use of robust and inexpensive matrices, and use of enzymes of either recombinant or plant origin.

Amino Acid Sequence↗

PubMed1985

Aminopeptidase N- and human blood group A-antigenicity along the digestive tract and associated glands in the rabbit.

The cellular localization of an aminopeptidase N homologous to the brush-border intestinal enzyme and that of human blood group A-substances were investigated using the immunofluorescence technique on thin frozen sections (200 nm) of the digestive tract and associated glands of A+ and A- rabbits. Aminopeptidase N was found to be a common specific marker of both the apical region of plasma membrane of acinar cells in submaxillary and parotid glands and pancreas and the brush border of jejunum and colon absorbing cells. In hepatocytes, the enzyme was localized in the sinusoidal domains. Soluble A-substances were present in mucus secretory granules of intestinal goblet cells and those of stomach and gall bladder mucous cells. In contrast, the mucous acini of sublingual and submaxillary glands were devoid of A-antigenicity. The columnar cells of striated ducts of these glands exhibited A-antigenicity. Soluble A-substances were also found in zymogen granules of parotid and pancreas acinar cells and those of stomach chief cells. Moreover, in all cells secreting A-substances, and in the non-secreting absorbing intestinal cells, the glycoproteins of the plasma membrane bore A-determinants. Aminopeptidase N was one of the membrane-bound glycoproteins that bore A-determinants in cells that expressed A-antigenicity.

ABO Blood-Group System↗

PubMed1981

Biochemical studies of aminopeptidase polymorphism in Mytilus edulis. II. Dependence of reaction rate on physical factors and enzyme concentration.

Enzymatic parameters of aminopeptidase-I that may be sensitive to temperature an solute variations were investigated to provide a functional explanation for specific activity differences among genotypes in natural populations. The effect of temperature on the apparent Km of L-leucyl-4-methoxy 2-naphthylamide and th dipeptide phenylalanyl-glycine was small, especially between 10 and 25 C. The apparent Km varied only between 36.7 and 49.8 microM at these temperatures and the six common genotypes did not differ in temperature-dependent substrate affinities. While pH had a significant effect on Km, no differences among genotypes were observed. Activation enthalpies were also identical among genotypes. Thermal inactivation was slowest at 15 C and the same for all genotypes. Of 18 tested amino acids, only phenylalanine inhibited aminopeptidase-I; K1 values ranged from 1.2 to 0.8 mM and were the same for all genotypes. Small differences among genotypes were detected in the inhibitory effect of zinc. The concentration of aminopeptidase-I enzyme was the same for all genotypes in a population exposed to oceanic salinity, but the concentration of Lap 94/94 was 15% lower than that of other genotypes in a population experiencing estuarine salinity. Genotypes with the Lap 94 allele exhibited higher apparent Kcat values in all population samples.The probable genotype-dependent effects of enzyme concentration and Kcat differences are discussed with regard to maintenance of the polymorphism and genetic differences among populations.

Aminopeptidases↗