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PubMed1993

Periplasmic aminopeptidases in Acinetobacter calcoaceticus and Pseudomonas aeruginosa.

The greater part of the intracellular aminopeptidases in Pseudomonas aeruginosa and Acinetobacter calcoaceticus is soluble. The localization of aminopeptidases in the cells was examined using the osmotic shock method with some modifications. When the cells of A. calcoaceticus and P. aeruginosa of the logarithmic phase were subjected to an osmotic shock, all aminopeptidases investigated were mainly localized in the sucrose supernatants and in the periplasm. Acid phosphatase as marker enzyme for periplasm showed a similar distribution between the fractions as the aminopeptidases. The periplasmic aminopeptidases of both microorganisms were separated by FPLC on Superose 12 and their molecular masses were determined. The results obtained show that at least four different aminopeptidases occur in the periplasm, a leucyl aminopeptidase (LAP, cleaving Leu-NH-NH2, 400 kDa), a glutamyl aminopeptidase (GAP, 200 kDa), an alanyl aminopeptidase (AAP, 80 kDa) and a prolyl aminopeptidase (PAP, 65 kDa). The results are in agreement for both species. Our results show clearly that aminopeptidases of these typical members of Gram-negative bacteria are mainly periplasmic like degrading enzymes (alkaline and acid phosphatases, 5'-nucleotidase, cyclic phosphodiesterase), detoxifying enzymes and binding proteins for amino acids and sugars.

Acinetobacter calcoaceticus↗

PubMed1990

Characterization of aminopeptidases in human kidney soluble fraction.

Fractionation of human kidney soluble extract (with which the majority of cellular aminopeptidase activity is associated) via anion exchange chromatography resolved four types of separable aminopeptidase (relative activity in parenthesis): alanyl aminopeptidase (EC 3.4.11.14; 50%); arginyl aminopeptidase (EC 3.4.11.6; 30%); leucyl aminopeptidase (EC 3.4.11.1; 18%) and pyroglutamyl aminopeptidase (EC 3.4.19.3; 2%). The further purification (via gel filtration chromatography and preparative electrophoresis) and characterization of each aminopeptidase has been described; the aminopeptidase tissue profile for human kidney was found to be similar to that previously obtained for human skeletal muscle and brain tissue using a similar experimental approach, i.e. the same enzymes, with corresponding similar characteristics, are present in each tissue. These results suggest that soluble aminopeptidases may be of fundamental importance in general cell protein catabolism. Degradation of the following aminoacyl-AMC derivatives via kidney soluble extract has been shown to be due principally to hydrolysis by alanyl aminopeptidase: glutamyl, glycyl, isoleucyl, methionyl, ornithyl, phenylalanyl, prolyl, seryl, tyrosyl and valyl. We would suggest that measurement of the soluble extract-derived aminopeptidases described in this paper in urine may lead to an improvement upon existing assay procedures for early detection of kidney damage.

Aminopeptidases↗

PubMed1981

Solubilization and characterization of two rat brain membrane-bound aminopeptidases active on Met-enkephalin.

Two aminopeptidases which hydrolyze Metenkephalin at the Tyr-Gly bond have been solubilized from rat brain membranes and resolved by ion-exchange chromatography. These aminopeptidase are designated MI and MII based on the order in which they are eluted during ion-exchange chromatography. The two aminopeptidases can be distinguished kinetically; aminopeptidase MI hydrolyzes L-arginine beta-naphthylamide 17 times faster than L-alanine beta-naphythylamide, while only a 1.7-fold difference is exhibited by aminopeptidase MII. Aminopeptidase MII exhibits a higher affinity for amino acid beta-naphthylamides, Met-enkephalin, Leu-enkephalin, and the inhibitor puromycin as compared to aminopeptidase MI. Greater than 90% of aminopeptidase MII activity is lost upon dialysis against ethylene-diaminetetraacetate (EDTA) but can be reconstituted with CoCl2 and MnCl2. In contrast, aminopeptidase MI loses only 30% of its activity when dialyzed against EDTA. In addition to cleaving the Tyr-Gly bond of Met-enkephalin, aminopeptidase MII also cleaves the Tyr-Gly bond of alpha- and gamma-endorphin. Hydrolysis of Met-enkephalin by intact membranes derived from whole rat brain occurs primarily by cleavage at the Tyr-Gly bond, with this activity attributable to aminopeptidase MII.

Aminopeptidases↗

PubMed1992

Identification and characterization of aminopeptidases from Aplysia californica.

Aminopeptidase activities were identified in extracts of kidney, ovotestis, head ganglia, heart and haemolymph of Aplysia californica. These enzyme preparations hydrolysed [3H][Leu]enkephalin at the Try-1-Gly-2 bond as determined by h.p.l.c. analysis of cleavage products. In all these tissues, enkephalin-degrading aminopeptidase activities were present both in membrane-bound and cytosolic fractions. The bivalent-cation-chelating agent, 1,10-phenanthroline, inhibited kidney membrane aminopeptidase activity with an IC50 of 30 microM, suggesting that this enzyme is a metalloproteinase. The aminopeptidase inhibitor amastatin was the most potent inhibitor of [Leu]enkephalin degradation (IC50 25 nM) by membrane-bound aminopeptidase, and bacitracin, bestatin and puromycin were about 100-1000 times less potent. In contrast with membrane-bound aminopeptidase, the cytosolic form is sensitive to puromycin. Angiotensin-converting enzyme inhibitor had no effect on [Leu]enkephalin degradation by kidney membranes, while the neutral endopeptidase inhibitors were poor inhibitors of the enzymes in this preparation. The Km values of the aminopeptidase in the kidney membranes and cytosolic fractions for the [Leu]enkephalin substrate were 2.4 and 7.4 microM respectively. The aminopeptidase present in the kidney membranes also hydrolysed endogenous Phe-Met-Arg-Phe-amide peptide at the Phe-1-Met-2 bond as well as synthetic alanine p-nitroanilide and leucine p-nitroanilide. When used in a competition assay, these substrates inhibited hydrolysis of [3H][Leu]enkephalin, suggesting that the same enzyme degraded all these substrates. Taken together, these results suggest that Aplysia tissues contain both a membrane-bound aminopeptidase related to the mammalian aminopeptidase N and a cytosolic puromycin-sensitive aminopeptidase.

Aminopeptidases↗

PubMedDate not supplied

Regional and subcellular distribution of soluble aminopeptidase in the human and the rat brain: a comparative study.

In an attempt to elucidate the cellular function of the soluble aminopeptidases, we have analysed their activity in several subcellular fractions (synaptosomal, mitochondrial, microsomal, nuclear and cytosolic fraction) and in different areas (amygdala, hypothalamus, hippocampus, striatum, frontal cortex, occipital cortex and parietal cortex) of the human and the rat brain. The enzymes assayed in this study were five cytosolic aminopeptidases identified inmammalian brain tissues: alanyl-aminopeptidase, arginyl-aminopeptidase, leucyl-aminopeptidase, pyroglutamyl-peptidase I and aspartyl-aminopeptidase. The regional comparative study revealed significantly higher activities of alanyl-aminopeptidase activity in the human brain, with arginyl-aminopeptidase activities being higher in the rat brain. In the subcellular study, while the alanyl- and arginyl-aminopeptidase activities were quite homogeneous in all the subcellular fractions, the leucyl-aminopeptidase, pyroglutamyl-peptidase I and aspartyl-aminopeptidase activities were significantly higher in the synaptosomal fraction. The differential distribution of these enzymes could suggest that these activities have different functions in the distinct subcellular structures of the human and the rat brain.

Aminopeptidases↗

PubMed2005

Reversal of angiotensin II-stimulated collagen gel contraction in cardiac fibroblasts by aminopeptidase inhibition.

The purpose of this investigation was to determine whether aminopeptidase inhibition could affect the angiotensin II-stimulated collagen gel contraction in basal (control) and TGF-beta1-treated cardiac fibroblasts (or myofibroblasts). The tested aminopeptidase inhibitors were the broad range aminopeptidase inhibitor bestatin, the specific inhibitor of alanine aminopeptidase leuhistin, and the specific inhibitor of arginine aminopeptidase arphamenine A. Cardiac fibroblasts (from normal male adult rats) from passage 2 were cultured to confluency and incubated with(out) 400 pmol/L TGF-beta1 in Dulbecco Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). These fibroblasts were then further incubated in a floating collagen gel lattice with the tested products (angiotensin II, bestatin, leuhistin, or arphamenine A) for 3 days in DMEM without FBS. The contraction of the collagen gel lattice by cardiac fibroblasts was determined by measuring the gel volume with tritiated water. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline from alanine- or arginine-p-nitroanilide. Angiotensin II (100 nmol/L) reduced the gel volume in control and TGF-beta1-treated cardiac fibroblasts. The angiotensin II-stimulated collagen gel contraction in control and TGF-beta1-treated fibroblasts was almost completely reversed by leuhistin and arphamenine A (100 micromol/L). Bestatin (100 micromol/L) only partially inhibited the angiotensin II-stimulated collagen gel contraction in control fibroblasts, although it did not affect the angiotensin II-induced contraction in TGF-beta1-treated fibroblasts. In control and TGF-beta1-treated cardiac fibroblasts, 100 micromol/L leuhistin or arphamenine A only partially inhibited alanine aminopeptidase activity, whereas bestatin (100 micromol/L) completely inhibited the alanine aminopeptidase activity. Arginine aminopeptidase activity was only partially inhibited by leuhistin and arphamenine A at 100 micromol/L in control and TGF-beta1-treated fibroblasts. Bestatin, however, completely blocked the arginine aminopeptidase activity in control fibroblasts and only partially in TGF-beta1-treated fibroblasts at 100 micromol/L. Our data suggest that both alanine and arginine aminopeptidases are involved in the reversal of the angiotensin II-stimulated collagen gel contraction in control and TGF-beta1-treated cardiac fibroblasts or myofibroblasts.

Aminopeptidases↗

PubMed1977

Aminopeptidase N from Escherichia coli. Unusual interactions with the cell surface.

The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30-40%) by osmotic shock or by converting Escherichia coli K10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75-85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N-ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I-labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and beta-galactosidase to fluorescamine in intact cells. Only 16% of the total beta-galactosidase was labeled with this fluorescent reagent whereas 44-45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places. Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.

Alkaline Phosphatase↗

PubMed1985

The slow, tight binding of bestatin and amastatin to aminopeptidases.

Bestatin reversibly inhibits Aeromonas aminopeptidase (EC 3.4.11.10) in a process that is remarkable for its unusual degree of time dependence. The binding of bestatin by both Aeromonas aminopeptidase and cytosolic leucine aminopeptidase (EC 3.4.11.1) is slow and tight, with Ki values (determined from rate constants) of 1.8 X 10(-8) and 5.8 X 10(-10) M, respectively. In contrast, microsomal aminopeptidase (EC 3.4.11.2) binds bestatin in a rapidly reversible process with a Ki value of 1.4 X 10(-6) M. Kinetic analysis of the slow inhibition observed is facilitated by the use of a variety of experimental treatments, primarily measurements made during pre-equilibrium; however, careful selection of conditions permits use also of steady state observations. When titrated with bestatin, 1 mol of cytosolic leucine aminopeptidase (containing 6 g atoms each of zinc and manganese) is rendered 80% inactive by 1 mol of inhibitor, thus suggesting that enzymatic activity depends on one active site/hexamer; titration of Aeromonas aminopeptidase by bestatin reveals a 1:1 stoichiometry. Amastatin inhibits all three aminopeptidases through the mechanism of slow, tight binding with Ki values ranging from 3.0 X 10(-8) to 2.5 X 10(-10) M. This behavior of microsomal aminopeptidase contrasts sharply with its rapidly reversible inhibition by bestatin. The slow, tight binding observed with five of the six aminopeptidase-inhibitor pairs investigated suggests the formation of a transition state analog complex between the enzyme and inhibitor. Physical evidence consistent with this possibility was provided by the observation that both bestatin and amastatin perturb the absorption spectrum of cobalt Aeromonas aminopeptidase.

Aeromonas↗

PubMed1983

Characterization of three aminopeptidases purified from human placenta.

Three aminopeptidases purified from the human placenta were characterized and compared with each other. Aminopeptidase II1 preferred L-arginine- and L-lysine-beta-naphthylamides or p-nitroanilides as substrate, with low or negligible hydrolysis of other amino acid derivatives. It was inhibited by L-arginine, L-lysine and L-methionine. This enzyme activity was highly sensitive to heat treatment, N-ethylmaleimide, p-chloromercuribenzoate, puromycin, bestatin, epsilon-amino-n-caproic acid (EACA) and EDTA. After EDTA, this enzyme could be reactivated by Co2+. It is concluded that aminopeptidase II1 is identical with arginine aminopeptidase (EC 3.4.11.6) from other mammalian tissues. Aminopeptidase II2 preferred L-alanine-beta-naphthylamide and p-nitroanilide as substrates. It was also able to hydrolyse L-leucine, L-arginine, L-methionine and L-lysine derivatives but only very weakly L-cystine and Bz-L-cysteine substrates. This enzyme was inhibited by L-arginine, L-alanine, L-lysine and most strongly by L-leucine and L-methionine. It was resistant to bestatin and heat treatment but sensitive to EACA. EDTA caused a marked suppression, which could be prevented by Co2+ and Zn2+. These characteristics are reminiscent of those of alanine aminopeptidase (EC 3.4.11.-) found in other tissues. The third enzyme was the only one clearly particle bound and was therefore called PB-aminopeptidase. It preferred L-leucine derivatives as substrate but also readily hydrolysed other amino acid-beta-naphthylamides and p-nitroanilides including L-cystine and Bz-L-cysteine substrates. Among the amino acids L-cysteine, L-leucine and L-methionine were inhibitory. Bestatin and thiol reagents were without effect and EACA was only moderately inhibitory. EDTA caused a strong suppression, which could be prevented by Co2+ and Zn2+. These properties are equal to those previously described for the placental cystine aminopeptidase (oxytocinase) (EC 3.4.11.3). All three enzymes had an optimum close to neutral pH but apparent differences in their Km and Vmax values with various substrates. These findings suggest that the three purified aminopeptidases are distinct enzymes. Two of these (aminopeptidases II1 and II2) have not previously been isolated and characterized in the human placenta.

Amino Acids↗

PubMed2001

Pituitary aminopeptidase activities involved in blood-pressure regulation are modified by dietary cholesterol: sex differences.

Given that the existence of a local renin-angiotensin system (RAS) in the pituitary and its participation in the regulation of blood pressure and other biological functions are widely accepted, the aim of this work is to analyze the influence of dietary cholesterol on the activity of the enzymes involved in the metabolism of the effector peptides of the renin-angiotensin system (angiotensin II and III) and vasopressin, in the pituitary of male and female mice fed on a cholesterol-enriched diet (1% cholesterol and 0.5% cholic acid). Soluble and membrane-bound pituitary aminopeptidase A (aspartyl- and glutamyl-aminopeptidase), aminopeptidase M (alanyl-aminopeptidase), aminopeptidase B (arginyl-aminopeptidase) and cystinyl-aminopeptidase activities were fluorimetrically measured. In female mice, cholesterol-enriched diet produced a significant increase in soluble aspartyl- and membrane-bound aspartyl- and glutamyl-aminopeptidase activities, and a significant decrease in membrane-bound alanyl-, arginyl- and cystinyl-aminopeptidase activities. In male mice, after feeding the diet, a significant increase in soluble glutamyl- and membrane-bound arginyl-aminopeptidase activities was observed. Our results indicate differential effects of dietary cholesterol on the metabolism of angiotensin II and III and vasopressin in the pituitary of male and female mice.

Animals↗

PubMed1985

Properties and activities of aminopeptidases in normal and mitogen-stimulated human lymphocytes.

Human peripheral lymphocytes were found to contain at least two distinct aminopeptidases, designated cytosol aminopeptidase and microsomal aminopeptidase, which differed from one another with respect to intracellular localization, substrate specificity, metal-ion activation, Km value and electrophoretic mobility. No change in these aminopeptidase activities was observed in cultured lymphocytes in the absence of mitogen throughout the cultivation period. The addition of phytohaemagglutinin or concanavalin A to the culture medium caused, in dose-dependent manner, a significant increase in cytosol aminopeptidase activity in lymphocytes. On the other hand, no increase in microsomal aminopeptidase activity was observed under the same conditions. The biochemical properties of aminopeptidases in stimulated cultured lymphocytes were identical with those of the enzymes in peripheral lymphocytes and unstimulated cultured lymphocyte. The phytohaemagglutinin dose-response curves for lymphocyte activation as measured by the DNA synthesis rate and for cytosol aminopeptidase activity were observed to be similar. However, when DNA synthesis was temporarily blocked by hydroxyurea, the rate of increase of aminopeptidase activity was unaffected. Pokeweed mitogen only slightly increased the cytosol aminopeptidase activity in cultured lymphocytes, although the lymphocytes were highly activated.

Aminopeptidases↗

PubMed1985

The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase N.

The property of solutions of Triton X-114 to separate into detergent-rich and detergent-poor phases at 30 degrees C has been exploited to investigate the identities of the aminopeptidases in synaptic membrane preparations from pig striatum. When titrated with an antiserum to aminopeptidase N (EC 3.4.11.2), synaptic membranes solubilized with Triton X-100 revealed that this enzyme apparently comprises no more than 5% of the activity releasing tyrosine from [Leu]enkephalin. When assayed in the presence of puromycin, this proportion increased to 20%. Three integral membrane proteins were fractionated by phase separation in Triton X-114. Aminopeptidase activity, endopeptidase-24.11 and peptidyl dipeptidase A partitioned predominantly into the detergent-rich phase when kidney microvillar membranes were so treated. However, only 5.5% of synaptic membrane aminopeptidase activity partitioned into this phase, although the other peptidases behaved predictably. About half of the aminopeptidase activity in the detergent-rich phase could now be titrated with the antiserum, showing that aminopeptidase N is an integral membrane protein of this preparation. Three aminopeptidase inhibitors were investigated for their ability to discriminate between the different activities revealed by these experiments. Although amastatin was the most potent (IC50 = 5 X 10(-7) M) it failed to discriminate between pure kidney aminopeptidase N, the total activity of solubilized synaptic membranes and that in the Triton X-114-rich phase. Bestatin was slightly more potent for total activity (IC50 = 6.3 X 10(-6) M) than for the other two forms (IC50 = 1.6 X 10(-5) M). Puromycin was a weak inhibitor, but was more selective. The activity of solubilized membranes was more sensitive (IC50 = 1.6 X 10(-5) M) than that of the pure enzyme or the Triton X-114-rich phase (IC50 = 4 X 10(-4) M). We suggest that the puromycin-sensitive aminopeptidase activity that predominates in crude synaptic membrane preparations may be a cytosolic contaminant or peripheral membrane protein rather than an integral membrane component. Aminopeptidase N may contribute to the extracellular metabolism of enkephalin and other susceptible neuropeptides in the brain.

Aminopeptidases↗

PubMed1992

Leucine aminopeptidase from Arabidopsis thaliana. Molecular evidence for a phylogenetically conserved enzyme of protein turnover in higher plants.

Leucine aminopeptidases are exopeptidases which are presumably involved in the processing and regular turnover of intracellular proteins; however, their precise function in cellular metabolism remains to be established. Towards this goal, a full-length complementary DNA encoding a plant leucine aminopeptidase was isolated from a cDNA library of Arabidopsis thaliana and sequenced. The nucleotide sequence showed 49.5% identity to the Escherichia coli xerB-encoded leucine aminopeptidase. Sequence analysis revealed that the cDNA encodes a polypeptide of 520 amino acids with a calculated molecular mass of 54,506 Da. The C-terminal part (amino acids 200-520) of the deduced amino acid sequence showed 43.8% sequence identity to the xerB-encoded leucine aminopeptidase and 42.6% sequence identity to the amino acid sequence of bovine lens leucine aminopeptidase (EC 3.4.11.1). No sequence similarity (not even over short sequence elements) was observed with any other known peptidase or proteinase sequence. The cDNA was expressed as a fusion protein from the lacZ promoter in E. coli. Enzymatic analysis proved that the cloned cDNA encoded an active leucine aminopeptidase. The properties of this enzyme, including metal requirements, inhibitor sensitivity, pH optimum and the remarkable temperature stability, are very similar to those reported for leucine aminopeptidases from other tissues. Amino acids involved in metal and substrate binding in bovine lens aminopeptidase are completely conserved in the plant enzyme as well as in the XerB protein. Our results show that leucine aminopeptidases form a superfamily of highly conserved enzymes, spanning the evolutionary period from the bacteria to animals and higher plants. This is the first aminopeptidase cloned from a plant.

Amino Acid Sequence↗

PubMed2002

Denaturation of either Manduca sexta aminopeptidase N or Bacillus thuringiensis Cry1A toxins exposes binding epitopes hidden under nondenaturing conditions.

The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with (125)I-labeled Cry1Ac, Cry1Ac mutant (509)QNR-AAA(511) (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both (125)I-Cry1Ac and (125)I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots (125)I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, (125)I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. (125)I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured (125)I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) (125)I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.

Aminopeptidases↗

PubMed1996

Inhibition of tumor cell invasion and matrix degradation by aminopeptidase inhibitors.

We investigated the effects of several types of aminopeptidase inhibitors on tumor cell-associated aminopeptidase activity and invasion. The aminopeptidase expressed by the human metastatic HT1080 fibrosarcoma cells was effectively suppressed by actinonin A, bestatin, leuhistin and matlystatin A, which are capable of inhibiting the purified aminopeptidase N, but not by arphamenine B specific for aminopeptidase B. The aminopeptidase N inhibitors inhibited HT1080 cells from degrading the subendothelial matrix and from invading into Matrigel in parallel with their aminopeptidase inhibitory activities. Matlystatin A, with multiple inhibitory activity against both aminopeptidase N and matrix metalloproteinases (MMP), was the most effective inhibitor of invasion. However, leuhistin and bestatin, without MMP inhibitory activity, also exhibited significant inhibition of invasion. The results suggest that aminopeptidase N plays a crucial role in the degradation and invasion of extracellular matrices by fibrosarcoma cells and that aminopeptidase inhibitors may be useful for preventing the spread of malignant tumors.

Aminopeptidases↗

PubMed1984

Aminopeptidases from human leucocytes.

Six aminopeptidases differing in enzymic specificity against various L-amino acid-4-nitroanilides were detected and isolated from the cytosol of leucocytes collected from the buffy coat of human blood. The different enzymes were separated by one step of chromatography on DEAE-Sephacel and were further purified by gel filtration on Sephacryl S-300. The main aminopeptidases of the cytosol were designated aminopeptidases 1, 2, 4 and 5 (AP 1, AP 2, AP 4, AP 5) on the basis of their elution sequence from the first ion-exchange chromatography column on DEAE-Sephacel. Aminopeptidase 1 appeared to be a strongly sulfhydryl-dependent leucine aminopeptidase, activatable by thiol reagents. The enzyme was inhibited by p-chloromercuribenzoate. Its molecular mass was estimated to be 150 kDa. Aminopeptidase 2 showed high specificity for proline-4-nitroanilide. This enzyme was inhibited by p-chloro-mercuribenzoate and bestatin. It exhibited a molecular mass of 70 kDa. Aminopeptidase 4 designated the activities of two different enzymes of apparent molecular masses of 220 and 70 kDa which could be further separated by gel filtration. Aminopeptidase 5 exhibited the properties alike aminopeptidase B with high specific hydrolytic activity against the 4-nitroanilides of lysine and arginine. The molecular mass was estimated to be 90 kDa. Aminopeptidase 3 was a minor component in the cytosol and could be identified as an extracellular leucocyte plasma membrane constituent. The enzyme exhibited properties of a metallo proteinase and could be inhibited by EDTA. The molecular mass was estimated to be 250 kDa.

Aminopeptidases↗

PubMed1999

Intramolecular chaperone and inhibitor activities of a propeptide from a bacterial zinc aminopeptidase.

An aminopeptidase from Aeromonas caviae T-64 was translated as a preproprotein consisting of three domains; a signal peptide (19 amino acid residues), an N-terminal propeptide (101 residues) and a mature region (273 residues). We demonstrated that a proteinase, which was isolated from the culture filtrate of A. caviae T-64, activated the recombinant pro-aminopeptidase by removal of the majority of the propeptide. Using L-Leu-p-nitroanilide as a substrate, the processed aminopeptidase showed a large increase in kcat when compared with the unprocessed enzyme, whereas the Km value remained relatively unchanged. The similar Km values for the pro-aminopeptidase and the mature aminopeptidase indicated that the N-terminal propeptide of the pro-aminopeptidase did not influence the formation of the enzyme-substrate complex, suggesting the absence of marked conformational changes in the active domain. In contrast, the marked difference in kcat suggests a significant decrease in the energy of one or more of the transition states of the enzyme-substrate reaction coordinate. Moreover, we showed that the activity of the urea-denatured pro-aminopeptidase could be recovered by dialysis, whereas the activity of the urea-denatured mature aminopeptidase, which lacked the propeptide, could not. Further to this, the propeptide-deleted aminopeptidase formed an inclusion body in the cytoplasmic space in Escherichia coli and was not secreted at all. These results suggested that the propeptide of the pro-aminopeptidase acted as an intramolecular chaperone that was involved with the correct folding of the enzyme in vitro and was required for extracellular secretion in E. coli.

Aeromonas↗

PubMedDate not supplied

Acetaldehyde inhibits serum aminopeptidases.

Aminopeptidase A (APA)- and aminopeptidase M (APM)-like activity were assayed in Moni-Trol ES with L-alpha-aspartyl-beta-naphthylamide and L-alanyl-beta-naphthylamide, respectively. Upon preincubation of the serum with 89.4, 223.5, and 447 mM acetaldehyde at room temperature for 30 min, a reduction in 26.8%, 55.3%, and 75.8% aminopeptidase A activity was observed. Similarly, aminopeptidase M activity was reduced by 26.5% and 53.1% upon preincubation with 223.5 and 447 mM acetaldehyde. Ethanol at 84.9, 212.3, and 427.9 mM did not significantly affect the enzymic activity. Because aminopeptidase A and aminopeptidase M also degrade the pressor substance, angiotensin II, it is suggested that inhibition of aminopeptidase A- and aminopeptidase M-like activity by acetaldehyde, the product of ethanol metabolism, may lead to higher levels of circulating angiotensin II and, consequently, hypertension, in alcoholics. The hydrolysis of lysine-p-nitroanilide, an aminopeptidase B substrate, was also inhibited upon addition of acetaldehyde to Moni-Trol ES serum.

Acetaldehyde↗