Aminopeptidase Substrates Research Articles

Cloning and Characterization of a Leucyl Aminopeptidase from Three Pathogenic Leishmania Species

Aminopeptidases are emerging as exciting novel drug targets and vaccine candidates in parasitic infections. In this study, we describe for the first time an aminopeptidase from three highly pathogenic Leishmania species. Intronless genes encoding a leucyl aminopeptidase (lap) were cloned from Leishmania amazonensis, Leishmania donovani, and Leishmania major, which encoded 60-kDa proteins that displayed homology to leucyl aminopeptidases from Gram-negative bacteria, plants, and mammals. The lap genes were present as a single copy in each genome, and lap mRNA was detected by reverse transcription-PCR in all life-cycle stages of L. amazonensis. Lap assembled into catalytically competent 360-kDa hexamers and demonstrated potent amidolytic activity against synthetic aminopeptidase substrates containing leucine, methionine, and cysteine residues, representing the most restricted substrate specificity of any leucyl aminopeptidase described to date. Optimal activity was observed against L-leucyl-7-amido-4-methylcoumarin (k(cat)/K(m) approximately 63 s(-1) x mm(-1)) with a pH optimum of 8.5. Leishmania Lap activity was inhibited by metal ion chelators and enhanced by divalent manganese, cobalt, and nickel cations, although only zinc was detected in the purified Lap by inductively coupled plasma atomic emission spectroscopy, indicating that zinc is the natural Lap cofactor. Activity was potently inhibited by bestatin and apstatin in a slow binding competitive fashion, with K(i)* values of 3 and 44 nm, respectively. Actinonin was a tight binding competitive inhibitor (K(i) approximately 1 nm), whereas arphamenine A (K(i) approximately 70 microm) and L-leucinol (K(i) approximately 100 microm) were non-tight binding competitive inhibitors. Lap was not secreted by Leishmania in vitro and was localized to the parasite cytosol.

Evaluation of methods to solubilize and analyze cell-associated ectoenzymes

To quantify the functional diversity of microbes that use hydrolytic ectoenzymes, the feasibility of separating cell-associated proteins on polyacrylamide gels and detecting enzyme activity via fluorescent substrate analogs for aminopeptidases, glucosidases, and esterases was determined. More than 87% of aminopeptidase activity was associated with particulate cell material in all of the 10 Gram-negative bacterial strains that were investigated. Although 7-amino-4-methylcoumarin-leucine (AMC-leucine) provided high activities after incubation with eight strains from the Cytophaga-Flexibacter-Bacteroides group, very poor responses were noted with two gamma Proteobacteria that grew well on protein. Therefore, this molecule was not a universal substrate for aminopeptidases. Methods of increasing stringency were evaluated to release enzyme activity from particulate material. Some methods (treatment with 0.1% Triton X-100) gave good results with some but not all strains. Cell lysis by shearing produced the most consistent results. Ectoenzyme activities could be localized on polyacrylamide gels using fluorescent substrate analogs. However, some activities were located in high molecular weight complexes, and methods that disrupted these complexes (such as treatment with sodium dodecyl sulfate at elevated temperature) destroyed enzyme activity. In addition, the enzymes from different strains showed the same electrophoretic mobility. Therefore, the analysis of functional diversity by this approach is limited by the difficulty in solubilizing particulate enzymes under conditions where they retain activity.

Tetrahydroisoquinoline derivatives of enkephalins: synthesis and properties

Tetrahydroisoquinolines (TIQs) are endogenous alkaloid compounds deriving from the non-enzymatic Pictet–Spengler condensation of catecholamines with aldehydes. These compounds are able to unsettle catecholamines uptake and release from synaptosomes and have been detected in urine and in post-mortem Parkinsonian brains. We have obtained in vitro, by the reaction of dopa-enkephalin (dopa-Gly–Gly–Phe–Leu) with acetaldehyde in the presence of rameic ions, a TIQ derivative of Leu-enkephalin. The isolation and the recovery of the peptide was obtained by HPLC. The acid hydrolysis and the subsequent analysis of the peptide lysate by the Amino acid analyser clearly revealed the absence of dopa, while the electrospray ionisation mass spectrometry showed that the sequence of the enkephalin derivative was the following: 3-carboxy-salsolinol-Gly–Gly–Phe–Leu (TIQ-enkephalin). This compound was not a good substrate for microsomal aminopeptidase and pronase with respect to Leu-enkephalin. Tested in the binding assay, the TIQ-enkephalin exhibited a very poor affinity toward the enkephalin receptors. When the TIQ-enkephalin was incubated with tyrosinase or peroxidase/H 2O 2, the formation of TIQ-opio-melanins occurred.

Protease Activity and Partial Characterization of the Trypsin-Like Enzyme in the Digestive Tract of the Tropical Sierra Scomberomorus concolor

Protease and trypsin-like activities of the digestive system of the tropical sierra Scomberomorus concolorwere evaluated. Sierra digestive tract extracts hydrolyzed specific substrates for trypsin, chymotiypsin, and leucine aminopeptidase At least eight bands of activity were observed by using SDS-PAGE. In PAGE and serineinhibition assays, one fraction resembled bovine trypsin. An ammonium sulfate (40-60%) trypsin-like fraction displayed different inhibitions with tosyllyschloromethyl ketone (96%) and soybean trypsin inhibitor (100%). Maximum activity was found using benzoyl-L-arginine-p-nitroanilide as substrate at pH 9.0 at 25°C and around 50°C at pH 7.8. The capacity of the trypsin-like fraction to work at different pHs (4, 7, and 9) at 25°C and different temperatures (0 and 40°C) at pH 7.8 were detected.

The role of aminopeptidases in haemoglobin degradation in Plasmodium falciparum-infected erythrocytes

Intra-erythrocytic Plasmodium parasites digest host cell haemoglobin and use the liberated amino acids for protein synthesis. Although several endoproteases (aspartic, cysteine, and metallo-) have been shown to be involved in the initial stages of haemoglobin degradation, little is known about the steps immediately before amino acid release. In our studies, fluorometric enzyme assays indicated that the stage of the P. falciparum erythrocytic cycle with highest aminopeptidase activity was the stage at which most haemoglobin degradation occurs, i.e. the trophozoite. Consistent with these results, metabolic growth assays indicated that the late ring/trophozoite stage was most susceptible to aminopeptidase inhibitors. To reconstitute the terminal stages of haemoglobin breakdown in vitro, we synthesised three peptides with amino acid sequences corresponding to known products of the endoproteolytic digestion of haemoglobin and employed them as substrates for aminopeptidases. Both trophozoite cytosolic extract, and partially-purified aminopeptidase, hydrolysed these peptide fragments to amino acids. Hydrolysis appeared to occur sequentially from the amino-termini of the peptides, and was inhibited in a concentration-dependent manner by the aminopeptidase-specific inhibitor nitrobestatin. The results suggest that P. falciparum aminopeptidases could be the enzymes responsible for the hydrolysis of haemoglobin-derived peptides to free amino acids. Lack of ultrastructural change in parasites treated with relevant concentrations of aminopeptidase-specific inhibitors, however, indicated that little feedback exists whereby the inhibition of cytosolic aminopeptidases results in obvious inhibition of initial haemoglobin degradation in the digestive vacuole.

Studies on the aminopeptidase activities of Porphyromonas gingivalis.

Porphyromonas gingivalis is an asaccharolytic bacterium that requires nitrogen substrates as carbon and energy sources. The aims of this study were to investigate the aminopeptidase activities of P. gingivalis and to evaluate the effect of aminopeptidase inhibitors on bacterial growth. Only arginine aminopeptidase and dipeptidyl aminopeptidase IV activities were detected. Experimental evidence was obtained suggesting that the Arg-gingipains of P. gingivalis can function as both an endopeptidase and an aminopeptidase. Firstly, the arginine aminopeptidase activity was found to be inhibited by leupeptin, a well-known inhibitor of Arg-gingipain activity. Secondly, a preparation of Arg-gingipain activity could hydrolyze the chromogenic substrate for arginine aminopeptidase. Lastly, a mutant of P. gingivalis constructed via gene disruption by use of suicide plasmids and deficient in both Arg-gingipain A and B was also devoid of arginine aminopeptidase activity. To investigate the key role of aminopeptidase activities in growth of P. gingivalis, aminopeptidase inhibitors were incorporated in the culture medium prior to inoculation. Bestatin and actinonin were the only ones to inhibit growth of P. gingivalis. Their mechanism of growth inhibition appears to be different but does not involve inhibition of the two major aminopeptidase activities (arginine aminopeptidase and dipeptidyl aminopeptidase IV).

Peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in Escherichia coli cell homogenates and intact cells.

An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.

L-Tyrosine β-naphthylamide is a potent competitive inhibitor of tyramine N-(hydroxycinnamoyl)transferase in vitro

l-Tyrosine β-naphthylamide, a synthetic substrate designed to measure tyrosine aminopeptidase activity, is a potent inhibitor of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) purified from elicited tobacco cell-suspension cultures. The inhibition is competitive, with the inhibitor binding reversibly to the tyramine binding site of the enzyme. Similar results were obtained with THT extracted from elicited potato cell-suspension cultures. K i values were found to be 0.66 μM for the enzyme from tobacco and 0.3 μM for the enzyme from potato. l-Tyrosine 7-amido-4-methylcoumarin, a fluorogenic substrate for tyrosine aminopeptidases, the structure of which is close to that of l-tyrosine β-naphthylamide, was also a powerful inhibitor, but slightly less effective with K i values of 0.72 and 0.42 μM for tobacco and potato THT, respectively. l-Tyrosine β-naphthylamide was rapidly hydrolysed when fed in vivo to tobacco or potato cell cultures or when incubated in crude enzymic extracts prepared from these cultures. This hydrolysis, which is presumably catalysed by aminopeptidases, precludes the use of l-tyrosine amides as inhibitors of THT in vivo.

Purification and characterization of aminopeptidase B from Escherichia coli K-12.

Aminopeptidase B, which is one of the four cysteinylglycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni2+, Mn2+, Co2+, and Cd2+, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidsase B is a metallopeptidase. It was stabilized against heat in the presence of Mn2+ or Co2+. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. Alpha-glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The kcat/Km for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.

Ethylene glycol and amino acid derivatives of 5-aminolevulinic acid as new photosensitizing precursors of protoporphyrin IX in cells.

Protoporphyrin IX (PpIX) is used as a photosensitizing agent in photodynamic detection and therapy (PDT) of cancer and is synthesized intracellularly from aminolevulinic acid (ALA) precursors. To evaluate means to specifically target ALA derivatives to defined cells, we have synthesized and characterized ethylene glycol esters and amino acid pseudodipeptide derivatives of ALA as potential specific substrates for cellular esterases and aminopeptidases, respectively. The PpIX formation induced by these products was investigated using cultures of human and rat cell lines of carcinoma and endothelial origins. The cytotoxicity of these compounds in the absence of light was also controlled. The results have shown that ethylenglycol esters can induce high levels of PpIX and are useful at concentrations below their cytotoxicity threshold. From the ALA-amino acid derivatives which were evaluated, the highest PpIX production was obtained using ALA derivatives of neutral amino acids, as compared to acidic or basic amino acids.

Fluorophores at the N Terminus of Nascent Chloramphenicol Acetyltransferase Peptides Affect Translation and Movement through the Ribosome

Structurally different fluorescent probes were covalently attached to methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f). All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 x 11 A, caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNA(f) compared with fMet-tRNA(f) with pyrene having the least and eosin the biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA.

Localization and characterization of soluble and plasma membrane aminopeptidase activities in Arabidopsis seedlings

We previously demonstrated that N,1-naphthylphthalamic acid is hydrolyzed at the root-hypocotyl transition zone and other regions of Arabidopsis thaliana seedlings, and that this reaction, like NPA-induced growth inhibition, is strongly promoted by blue light. In addition, NPA amidase activity was detected in plasma membrane-enriched fractions obtained from Arabidopsis seedlings. To further investigate this phenomenon, we tested the hypothesis that the arylamidase(s) responsible for NPA hydrolysis may also have aminopeptidase activity. The responses of Arabidopsis seedlings to various aminopeptidase substrates were tested. The hydrolysis of Tyr-, Trp-, Pro- and Gly-Pro-β-naphthylamide aminopeptidase substrates was shown to be histochemically localized at the root-hypocotyl transition zone and other regions where NPA hydrolysis also occurs. Blue light stimulated the in vivo activity of Tyr- and Pro-aminopeptidase activities, and far-red light stimulated the activity of the Trp-aminopeptidase. These same substrates also induced NPA-like growth inhibitory effects. In parallel experiments, aminopeptidase activities were detected in the supernatant and plasma membrane fractions of seedling extracts. The soluble AP activities resemble previously described neutral aminopeptidases with specificity for aromatic residues. The plasma membrane fraction hydrolyzed Tyr-, Trp-, Ala-Pro- and Pro-AP substrates, and also exhibited activity against Phe- and Leu-substrates. Many of the properties of the aminopeptidases, such as pH optima, metal requirements and responses to inhibitors, overlap with those of the previously characterized NPA amidase, suggesting that the latter may represent the combined activities of multiple aminopeptidases.

Localisation of enzymes in live spermatozoa by CellProbe reagents (preliminary results).

As a new approach, various synthetic fluorogenic substrates, the CellProbe reagents, were applied to examine the topography of their cleavage in vital human spermatozoa. These substrates are able to enter the cells without requiring previous cell permeabilization and can produce a fluorescent dye after cleavage, depending on enzyme activity. Vital spermatozoa from samples with normal spermiogram parameters showed fluorescence in different areas and intensity after incubation with a variety of substrates for aminopeptidase A, peroxides, subtilisin, dipeptidylpeptidase IV (DPP IV), cathepsin D, glucosidase and glucuronidase, but not with the substrate for galactosidase. Fluorescence was mainly located in the acrosomal cap (substrates for DPP IV, subtilisin, cathepsin D, glucosidase and glucuronidase) in the middle piece and head (substrates for peroxides, glucosidase), in the sperm head (substrates for aminopeptidase A) and occasionally in the tail (substrate for glucosidase). The substrate for subtilisin may play a role in andrology, because subtilisin is a serine protease like acrosin. This substrate may possibly be used to determine the acrosin activity in vital spermatozoa. The CellProbe reagents for fluorescence cytoenzymology may serve advanced methods in both clinical andrology and spermiological research, presuming that the characteristics and qualities of the synthetic substrates are correct. Therefore, more extended studies will be necessary to determine their clinical utility and significance under physiological and pathological conditions.

Chromatographic separation of fluorescent thiol adducts of 4-chloro-7-sulphobenzofurazan: Use as substrates for enzymes of the mercapturic acid xenobiotic pathway

Fluorescent adducts of 4-chloro-7-sulphobenzofurazan with cysteine, cysteinylglycine, reduced glutathione and N-acetylcysteine were prepared. Adducts were separated by HPLC on a 3-mm Nova-Pak C 18 reversed-phase column using isocratic elution with a solvent of acetonitrile–0.15 M phosphoric acid (5:95) buffered at pH 2.5. The adducts were detected using a fluorescence detector set at an excitation wavelength of 365 nm and an emission wavelength of 510 nm and an ultraviolet detector at 254 nm. The adduct of reduced glutathione was also formed by the action of the enzyme glutathione- S-transferase. This adduct acted as a substrate for the enzyme γ-glutamyltranspeptidase and the product of this reaction, the 4-chloro-7-sulphobenzofurazanyl derivative of cysteinylglycine, acted as a substrate for either dipeptidase or aminopeptidase M. The sequential enzymic effects could be detected by changes in the relative fluorescence intensity of the solutions to which the respective enzymes had been added but were more appropriately followed by changes in the HPLC elution profiles after enzymic treatment of solutions.

Nasal delivery of peptides: an in vitro cell culture model for the investigation of transport and metabolism in human nasal epithelium

We investigated the transport- and metabolism properties of three peptides in monolayers of human nasal epithelial cells. The effective permeability coefficients of thyrotropin-releasing hormone, met-enkephalin and human recombinant insulin were found to be 4.5, 4.4 and 0.4·10 −7 cm/s, respectively. The permeability was inversely proportional to the molecular weight and one order of magnitude lower than in excised nasal mucosa of rabbits. The metabolic cleavage of thyrotropin-releasing hormone (TRH) to the free acid by cytosolic prolyl-endopeptidase was also detected in human nasal cell monolayers, suggesting that ca. 10% of the total amount of TRH is transported via a transcellular pathway. Met-enkephalin is a substrate for aminopeptidases, located on the apical membrane of nasal epithelial cells. Metabolites and enzyme activity are comparable with literature data. Our studies demonstrate that not only morphological, but also functional properties of human nasal epithelial cells are preserved under in vitro conditions. Such a cell culture model based on human nasal cells could be beneficial for the characterization of peptide transport on a cellular level and for investigation of the absorption enhancer mechanism. Further studies are necessary, however, to establish correlations between in vitro permeabilities in cell cultures and nasal drug absorption in animals and humans.

Degradation of Vitellogenins by 170 kDa Trypsin-Like Protease in the Plasma of the Tilapia, Oreochromis niloticus

Proteolytic degradation of plasma vitellogenins during purification procedure has been noted in several teleost fishes. We have characterized here a trypsin-like serine protease in the plasma of the tilapia, Oreochromis niloticus, which degrades vitellogenins. The molecular mass of the protease was estimated as 230 kDa by gel filtration and as 170 kDa both by nondenaturing and by SDS-polyacrylamide gel electrophoresis. The protease efficiently hydrolyzed the synthetic peptide substrates for trypsin-like proteases but not the substrates for chymotrypsin-like proteases nor aminopeptidases. Hydrolysis of the peptide substrates was strongly inhibited by leupeptin, aprotinin and N-tosyl-L-lysine chloromethyl ketone and to a certain extent by chymostatin, 3,4-dichloroisocoumarin, phenylmethanesulfonyl fluoride, and soybean trypsin inhibitor. Leupeptin and aprotinin also inhibited the degradation of a vitellogenin in the plasma. Although the physiological functions of the 170 kDa protease in vivo have not been elucidated, the results on enzymatic properties of this protease will be useful for the isolation and characterization of vitellogenin not only in tilapia but also in other organisms.

Ubenimex (bestatin) inhibits invasion and matrilysin activation of human uterine cervical adenocarcinoma OMC-4 cells

The purpose of the study was to investigate the effect of the aminopeptidase inhibitor ubenimex (bestatin) on the invasive activity of cultured human uterine cervical adenocarcinoma cells. The enzymatic degradation of the extracellular matrix by tumor cells during the invasive process was also examined. The invasion of cervical adenocarcinoma OMC-4 cells into reconstituted basement membrane (Matrigel) was inhi-bited by the presence of bestatin in a concentration-dependent manner. However, bestatin did not have any effect on tumor cell proliferation and migration. The casein zymography of tumor conditioned medium showed that the treatment of tumor cells with bestatin resulted in the drastic reduction of the 19 kDa-enzyme level (matrilysin, active form) and the slight reduction of the 28 kDa-enzyme level (matrilysin, latent form) in OMC-4 cells. The effect of bestatin on matrilysin secreted by OMC-4 cells were also confirmed by immunoblot analysis, and the disappearance of matrilysin in the active form was found. Bestatin inhibited hydrolysing activities towards substrates of aminopeptidases in tumor cells, but did not directly inhibit matrilysin. These results suggest that bestatin may inhibit the invasion of uterine cervical adenocarcinoma OMC-4 cells possibly through the inhibitory mechanisms for production and activation of matrilysin modulated by tumor aminopeptidases.

Aminopeptidase-like Protease Released from Oocytes Affects Oocyte Surfaces and Suppresses the Acrosome Reaction in Establishment of Polyspermy Block in Oocytes of the MusselMytilus edulis

Suppression of the acrosome reaction of sperm on fertilized oocytes inhibits sperm–oocyte binding and is considered one of the three mechanisms responsible for polyspermy block in oocytes of the musselMytilus edulis(Togoet al.,1995). When unfertilized oocytes were inseminated in the presence of aminopeptidase inhibitors and the fertilized oocytes were inseminated again, neither the acrosome reaction nor sperm binding to fertilized oocytes were suppressed, suggesting that aminopeptidase-like protease participates in suppression of the acrosome reaction. The supernatant solution obtained after centrifuging a suspension of fertilized oocytes hydrolyzed aminopeptidase substrates, and these activities were inhibited strongly or effectively by aminopeptidase inhibitors. When unfertilized oocytes were incubated with this supernatant solution and inseminated, both the number of sperm bound and the acrosome reaction rate decreased, and these effects were reversed by conducting this treatment in the presence of aminopeptidase inhibitors. These results suggest that aminopeptidase-like protease released from the oocyte at fertilization affects the oocyte surface to suppress the acrosome reaction and consequently inhibits sperm–oocyte binding.

Peptidases of the Rumen Bacterium,Prevotella ruminicola

Prevotella(formerlyBacteroides) ruminicolais a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated thatP. ruminicolahad the greatest range and specific activity of dipeptidyl peptidases among the species tested.Streptococcus bovishydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, includingRuminobacter amylophilus, Ruminococcusspp. andVeillonella parvula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids inP. ruminicola. Ion-exchange chromatography of sonicated extracts ofP. ruminicolaM384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Ala4and Ala5; another was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro-4-methoxynapthylamide and was similar to dipeptidyl peptidase type IV XDPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. All of the enzymes, and particularly those active against Ala2-p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures.

Inhibition of aminopeptidase A activity causes an acute albuminuria in mice: an angiotensin II-mediated effect?

The hydrolase aminopeptidase A is an important regulator of the renin-angiotensin system, since it inactivates its most vasoactive component angiotensin II (Ang II). A single i.v. injection of a monoclonal antibody against mouse aminopeptidase A (ASD-4) induces a membranous-like glomerulonephritis in mice, characterized by an acute albuminuria, that is not dependent on complement, the coagulation system, or inflammatory cells. We hypothesized that this albuminuria is the consequence of a reduction in aminopeptidase A enzyme activity, that might subsequently lead to an increase in Ang II levels. Aminopeptidase A enzyme activity was analysed in vitro by a fluorimetric enzyme assay and in vivo by enzyme histochemistry. The role of Ang II in the induction of albuminuria in this model was studied by measuring the renal aminopeptidase A mRNA expression in our model by a competitive PCR assay as an indirect measure of Ang II levels. In addition, the role of Ang II in this model was studied by preventing the formation of Ang II with the angiotensin-converting enzyme inhibitor enalapril or by blocking of the Ang II receptor with the AT1 receptor antagonist losartan. Only antibodies that were able to inhibit the aminopeptidase A enzyme activity in vitro and in vivo induced an acute albuminuria in mice. Renal aminopeptidase A mRNA expression was increased by injection of the anti-aminopeptidase A antibody. Both enalapril and losartan treatment reduced the acute albuminuria, measured 1 day after injection of a monoclonal antibody against aminopeptidase A, by 91% and 83%, respectively. It is concluded that the induction of acute albuminuria is correlated to the enzyme-inhibiting capacity of the anti-aminopeptidase A antibodies. This impaired enzymatic activity most likely leads to an increase in the levels of Ang II, the best known substrate of aminopeptidase A. The results of our additional experiments are in keeping with our hypothesis that Ang II mediates this acute albuminuria. Whether this occurs by an increase of blood pressure or by a growth factor-like effect remains to be defined by further studies in this model.