In vivo effects of indomethacin on the activity of metal-containing enzymes.

1. Indomethacin injected subcutaneously at a single "ulcerogenic" dose decreased aminooxidase and leucine aminopeptidase activity and did not change alcohol dehydrogenase and ceruloplasmin activity. 2. Indomethacin administered at an oral "therapeutic" dose inhibited aminooxidase activity in small intestinal mucosa but not in liver and did not change leucine aminopeptidase activity in blood, liver and intestinal mucosa; it, however, increased alcohol dehydrogenase and ceruloplasmin activity. 3. The decreased activity of ceruloplasmin and alcohol dehydrogenase by metal deficiency increased after oral indomethacin treatment, reaching the control values when indomethacin was chelated with copper. 4. The results suggest the participation of endogenous metals in the indomethacin effect.     

Comparison of vaginal aminopeptidase enzymatic activities in various animals and in humans.

The specific enzymatic activity of four different aminopeptidases (aminopeptidase N, leucine aminopeptidase, aminopeptidase A and aminopeptidase B) in vaginal homogenates from rabbit, rat, guinea-pig, sheep and humans was compared. The purpose of the study was to find an appropriate animal model that can be used in degradation studies of protein and peptide drugs. Different substrates were used as the relative specific substrates for the determination of aminopeptidase enzymatic activity: 4-methoxy-2-naphthylamide of L-alanine for aminopeptidase N, 4-methoxy-2-naphthylamide of L-leucine for leucine aminopeptidase, 4-methoxy-2-naphthylamide of L-glutamic acid for aminopeptidase A and 4-methoxy-2-naphthylamide of L-arginine for aminopeptidase B. The vaginal aminopeptidase enzymatic activity of different species was determined spectrofluorometrically. The inhibition of aminopeptidase activity in the presence of bestatin and puromycin inhibitors was also investigated. The results showed the presence of aminopeptidase enzymatic activity in all vaginal homogenates in the order: sheep > guinea-pig > rabbit > or = human > or = rat. Based on the results of the hydrolysis and inhibition of the 4-methoxy-2-naphthylamide substrates, it was difficult to have an exact decision on the aminopeptidase type in the vaginal homogenates from the species studied. It was found that the aminopeptidase activity in rat, rabbit and humans was not statistically different. Therefore, we suggest that rats and rabbits could be used as model animals for vaginal enzymatic activity studies and for determination of the degradation of protein and peptide drugs in the vagina.     

Studies of platinum complex inhibition of leucine aminopeptidase.

The inhibition of swine kidney leucine aminopeptidase by N-alkyl-substituted etyhlenediamine liganded dihaloplatinum chelates has been investigated. The rate of this inhibition at 37 degrees is considerably less than that for the platinum tetra- and hexahalo complexes and also the ethylenediamine dihalo chelates. Only mixed inhibition was observed in these studies. For the time studies used here little if any inhibition occurred at room temperature. L-Methionine and L-Ala-L-Met did not reverse the platinum chelate inhibition nor did they prevent it.     

A comparative study on the rectal aminopeptidase enzymatic activities of different species

The aim of the present study was to compare the enzymatic activity of four different aminopeptidases (aminopeptidase N, leucine aminopeptidase, aminopeptidase A, aminopeptidase B) in rectal homogenates from different species: rabbit, rat, guinea-pig, sheep and human. Different substrates were used as the relative specific substrates for the determination of aminopeptidase enzymatic activity. For this purpose, 4-methoxy-2-naphthylamide of L-alanine for aminopeptidase N, 4-methoxy-2-naphthylamide of L-leucine for leucine aminopeptidase, 4-methoxy-2-naphthylamide of L-glutamic acid for aminopeptidase A and 4-methoxy-2-naphthylamide of L-arginine for aminopeptidase B were employed. The rectal aminopeptidase enzymatic activity was determined spectrofluorometrically. The inhibition of activity of aminopeptidase in the presence of bestatin and puromycin inhibitors was also investigated. The results showed the presence of aminopeptidase enzymatic activity in all rectal homogenates. Sheep and guinea-pig had the greatest aminopeptidase activity. The four aminopeptidase activities of rat and rabbit were not significantly different from each other. Human data was not evaluated statistically, due to insufficient sample. But the values of human data was close to those of the rabbit and rat values except for aminopeptidase A. Based on the data of the hydrolysis and inhibition of the 4-methoxy-2-naphthylamide substrates, it was rather difficult to determine the aminopeptidase type in the rectal homogenates of the species studied. It has been found that the aminopeptidase activities of rat and rabbit were not statistically different from each other and the human data were close to them.     

Leucine aminopeptidase activity in plasma of rats,after combined carboxy- and methemoglobinemia

After combined treatment of rats with 0.8 mmol NaNO₂/kg intraperitoneally and 2.4 mmol CO/kg subcutaneously the leucine aminopeptidase activity in plasma increased significantly. Doses of 0.8 mmol NaNO₂/kg or 2.4mmol CO/kg alone did not change the enzyme activity. Single injection of 9.6 mmol CO/kg also produced an increase of the leucine aminopeptidase activity in the plasma of both normal and adrenalectomized rats. The results support the assumption that the enhancement of leucine aminopeptidase activity after carbon monoxide poisoning is an oxygen deficiency effect, and not an effect which is mediated by the adrenals.The authors wish to thank Miss Dagmar Liedtke for skilful technical assistance.     

Effects of food dyes on Paramecium caudatum: toxicity and inhibitory effects on leucine aminopeptidase and acid phosphatase activity.

The toxic effect of 14 food dyes was studied in Paramecium caudatum. It was found that xanthene dyes containing halogen atoms in their molecules were more toxic than other groups of food dyes. Phloxin and rose bengal containing chlorine were especially toxic. The effect of food dyes on leucine aminopeptidase, acid phosphatase, and γ-glutamyl transpeptidase activity in P. caudatum was studied in order to investigate the mechanism of toxicity. Phloxin and rose bengal inhibited leucine aminopeptidase remarkably. The inhibitory effect of food dyes on leucine aminopeptidase in vitro is consistent with the toxic effect of the dyes on the survival time of P. caudatum. A possible correlation between toxicity and inhibition of the activity of enzymes involved in the digestive process is discussed.     

OF4949, new inhibitors of aminopeptidase B. IV. Effects of OF4949 and its derivatives on enzyme systems

OF4949-I and II inhibited aminopeptidase B from Ehrlich ascites carcinoma in a competitive way and the Ki value for both against L-arginine-beta-naphthylamide was 8 X 10(-9) M. Inhibition by I and II of various exopeptidases and endopeptidases was examined. OF4949-I and II both strongly inhibited leucine aminopeptidase and enkephalin-degrading aminopeptidase; I also inhibited enkephalinase B. The inhibitory effects of various derivatives of I and II on aminopeptidase B activity, showed that the terminal amino and carboxamide groups are essential for activity.     

Beta-phenyl cysteine: a leucine aminopeptidase inhibitor

Beta-phenyl cysteine: a leucine aminopeptidase inhibitor. The kinetic parameters of the inhibition of the leucine aminopeptidase by beta-phenyl cysteine are reported in this communication.     

INHIBITION BY BACITRACIN OF SOME HYDROLYTIC ENZYMES

Bacitracin was seen to inhibit noncompetitively the papain-catalyzed hydrolysis of N-benzoyl-DL-arginyl-2-naphthylamine and the hydrolysis of N-L-leucyl-2-naphthyl-amine catalyzed by subtilisin, papain, and leucine aminopeptidase. Alkaline phosphatase of E. coli, aminopeptidase B of rat liver, and trypsin were not affected to any noticeable extent. α-Chymotrypsin was only slightly inhibited. The bacitracin preparation studied was not found to contain zinc (when determined with an atomic absorption spectrophotometer). The values of K _i for the inhibition of papain, subtilisin, and leucine aminopeptidase were determined to be 5.0, 4.5, and 2.0 mM, respectively. The values of the maximum velocity, V, and the substrate constant, K _s, at different concentrations of bacitracin were calculated for trypsin, papain, subtilisin, and leucine amino-peptidase by the aid of a computer program using the Hanes' equation. Bacitracin also inhibited some aminopeptidase-like enzymes of human fetal liver, human oral fluid, and gingiva. The enzymes hydrolyzing N-L-leucyl-2-naphthylamine were the strongest inhibited and those hydrolyzing N-L-prolyl-2-naphthylamine the least inhibited. The selective effect of bacitracin was explained in terms of substrate specificity of the enzymes and in terms of different topology of enzyme surface around the active site. The enzymes which were inhibited more strongly (papain, subtilisin, and leucine aminopeptidase) were considered to be inhibited due to the “open” structure of their active centers.     

In vivo effect of tunicamycin on the expression of rat small intestinal brush border membrane glycoproteins and glycoenzymes

Tunicamycin, a known inhibitor of the lipid-dependent glycosylation of proteins, was used in vivo to study the biosynthesis of rat intestinal brush border membrane aminopeptidase N and dipeptidyl aminopeptidase IV. The incorporation of [3H]glucosamine into newly synthesized total protein of mucosal cell homogenates was inhibited by 60%, whereas incorporation of [3H]leucine was decreased only 21% by tunicamycin. This effect was much more pronounced in the brush border membrane fraction isolated from intestinal mucosal cells where incorporation of radiolabled leucine and glucosamine was reduced to 50 and 82% of control values respectively. An examination of the brush border membrane protein profile by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that there was a marked selective decrease in the amount of glycoproteins of molecular weights greater than 130 kD. In addition, there were decreased levels of assayable aminopeptidase N, dipeptidyl aminopeptidase IV and disaccharidase activity in intestinal mucosal cell homogenates and brush border membranes of tunicamycin-treated rats. Though tunicamycin decreased incorporation of newly synthesized aminopeptidase N and dipeptidyl aminopeptidase IV protein into brush border membranes by 70-75%, the newly synthesized enzyme that was incorporated was indistinguishable from that of controls. Further, non-glycoslyated forms of both enzymes were not detected in any other subcellular fractions. These results show that tunicamycin, an inhibitor of glycosylation, significantly affected the expression of brush border membrane glycoproteins, suggesting that both polypeptide synthesis and degradation of these proteins may be altered in the presence of this drug.     

Inhibition of aminopeptidases by peptides containing ketomethylene and hydroxyethylene amide bond replacements

Inhibitors of aminopeptidase enzymes have been prepared by the synthesis of peptide substrate analogues in which the scissile amide bond has been replaced with the hydrolytically stable ketomethylene (-COCH2-) and hydroxyethylene [-CH(OH)CH2-] functionalities. Two synthetic strategies were used to prepare the inhibitors, and the advantages and disadvantages of each are discussed. The synthesis of peptides that contain the hydroxyethylene isostere was complicated by competing lactone and lactam formation, and attempts to prepare free N-terminal dipeptide hydroxyethylene isostere derivatives were unsuccessful. All ketomethylene isosteres examined were weak inhibitors of both leucine aminopeptidase and aminopeptidase M. However, the ketomethylene inhibitor LysK(RS)Phe (58) (Ki = 4 nM) is a potent inhibitor comparable to the natural product, arphamenine A (ArgKPhe; Ki = 2.5 nM). Normal Michaelis-Menten kinetics for inhibition of membrane leucine aminopeptidase are observed in the absence of magnesium ion, but nonlinear kinetics were obtained in the presence of Mg2+.     

Processing of antigenic peptides by aminopeptidases

Antigenic peptides presented to major histocompatibility complex (MHC) class I molecules are generated in the cytosol during degradation of cellular proteins by the ubiquitin-proteasome proteolytic pathway. Proteasome can generate N-extended precursors as well as final epitopes, and then the precursors are processed to mature epitopes by aminopeptidases. Both cytosolic peptidases (i.e. puromycin-sensitive aminopeptidase, bleomycin hydrolase and interferon-gamma-inducible leucine aminopeptidase) and recently identified metallo-aminopeptidase located in the endoplasmic reticulum (i.e. adipocyte-derived leucine aminopeptidase/endoplasmic reticulum aminopeptidase 1 and leukocyte-derived arginine aminopeptidase) can generate final epitopes from precursor peptides. Some of these aminopeptidases are also considered to destroy certain antigenic peptides to limit the antigen presentation. Taken together, it is getting evident that aminopeptidases located in the cytosol and the lumen of endoplasmic reticulum play important roles in the generation of antigenic peptides presented to MHC class I molecules.     

Oral Absorption of Peptides: Influence of pH and Inhibitors on the Intestinal Hydrolysis of Leu-Enkephalin and Analogues

Leu-enkephalin (YGGFL) and several analogues were chosen as model peptides for the study of peptide absorption and hydrolysis in the rat jejunum. An HPLC assay was adapted to detect YGGFL or the analogues and metabolites. Peptide hydrolysis was studied in the rat jejunum using a single-pass perfusion method. Extensive hydrolysis of YGGFL was observed in the rat jejunum and approaches to reduce its metabolism were studied. The brush border enzymes are a major site of enkephalin hydrolysis. Lumenal peptidases were secondary to the brush border enzymes in hydrolyzing the enkephalins in this system. In the in situ perfusion system, YGGFL is hydrolyzed primarily to Tyr and GGFL by the brush border aminopeptidase and to YGG and FL by brush border endopeptidase. Lowering the jejunal pH below 5.0 significantly reduces aminopeptidase activity and, to a lesser extent, endopeptidase activity. An aminopeptidase inhibitor, amastatin, produced more pronounced inhibitory effects at higher pH and the endopeptidase inhibitors, tripeptides YGG and GGF, are effective even below pH 5.0. Coperfusion of YGGFL with a combination of aminopeptidase and endopeptidase inhibitors, e.g., amastatin and YGG, is more effective in inhibiting hydrolysis since both metabolic pathways are inhibited. Leu-D(Ala)²-enkephalin, while showing enhanced stability against aminopeptidase hydrolysis, is hydrolyzed at the Gly–Phe bond by the endopeptidase. Its hydrolysis is not affected by pH changes or amastatin but is decreased by YGG. The YGGFL wall permeability was estimated and is not a limiting factor for oral absorption.     

SOME IMMUNOCHEMICAL RELATIONSHIPS OF Aeromonas AMINOPEPTIDASE

Evidence for structural relationships between the aminopeptidase of Aeromonas proteolytica and several proteolytic enzymes, including two other aminopeptidases, was investigated by means of immunochemical techniques. The activity of Aeromonas aminopeptidase was strongly inhibited by its homologous antiserum (anti-AAP), which also diminished the activity of the endopeptidase of Aeromonas proteolytica, although substantially higher concentrations were required for the latter. In contrast, the aminopeptidase of Bacillus licheniformis was stimulated by incubation with anti-AAP, as were the activities of two types of Bacillus subtilis alkaline proteases. The activity of swine kidney leucine aminopeptidase and the aminopeptidase activity in pronase were unaffected by anti-AAP. The only evolutionary relationship suggested by the inhibitory and stimulatory reactions was that between the endopeptidase and the aminopeptidase of Aeromonas proteolytica.     

Synthesis of sulfur-containing analogues of bestatin. Inhibition of aminopeptidases by alpha-thiolbestatin analogues

Sulfur-containing amino acid and peptide analogues of bestatin [((2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl)-L-leucine] (1) have been synthesized and evaluated as inhibitors of aminopeptidase M (AP-M), leucine aminopeptidase (LAP), and aminopeptidase B (AP-B). The 2-thiolbestatin analogue (6) was found to be a potent inhibitor of all three aminopeptidases (AP-M, Ki = 4.4 microM; LAP, Ki = 0.55 microM; AP-B, Ki = 4.6 nM) but only a slightly better inhibitor of these aminopeptidases than the parent hydroxy-containing compound 1. Synthetic analogues of L-leucinethiol(4), a strong inhibitor of aminopeptidases, were prepared in which the carbon alpha to the thiol groups was substituted with methyl, methyl carboxylate, and carboxamide derivatives and found to be much weaker inhibitors of all aminopeptidases. A thioamide analogue of bestatin (49) is a modest inhibitor of AP-M (Ki = 40 microM), LAP (Ki = 0.33 microM), and AP-B (Ki = 2.4 microM). These results suggest that the sulfur atoms in 2-thiolbestatin and bestatin thioamide do not interact strongly with the active-site zinc atom of these aminopeptidases when the inhibitors are bound to the enzyme. These results are not consistent with proposed models for the inhibition of aminopeptidases by bestatin and related analogues.     

K-13, a novel inhibitor of angiotensin I converting enzyme produced by Micromonospora halophytica subsp. exilisia. I. Fermentation, isolation and biological properties

A novel inhibitor of angiotensin I converting enzyme (ACE), designated K-13, was isolated from the culture broth of Micromonospora halophytica subsp. exilisia K-13. K-13 inhibited ACE non-competitively when hippuryl-L-histidyl-L-leucine was used as a substrate. The inhibition constant (Ki) was 0.349 microM. K-13 hardly inhibited carboxypeptidase A, trypsin, alpha-chymotrypsin, leucine aminopeptidase, and aminopeptidase B even at a level of 61 microM. When K-13 was administered intravenously to rats, it inhibited the pressor response to angiotensin I.     

The most potent organophosphorus inhibitors of leucine aminopeptidase. Structure-based design,chemistry, and activity

A new class of very potent inhibitors of cytosol leucine aminopeptidase (LAP), a member of the metalloprotease family, is described. The X-ray structure of bovine lens leucine aminopeptidase complexed with the phosphonic acid analogue of leucine (LeuP) was used for structure-based design of novel LAP inhibitors and for the analysis of their interactions with the enzyme binding site. The inhibitors were designed by modification of phosphonic group in the LeuP structure toward finding the substituents bound at the S' side of the enzyme. This resulted in two classes of compounds, the phosphonamidate and phosphinate dipeptide analogues, which were synthesized and evaluated as inhibitors of the enzyme. The in vitro kinetic studies for the phosphinate dipeptide analogues revealed that these compounds belong to the group of the most effective LAP inhibitors found so far. Their further modification at the P1 position resulted in more active inhibitors, hPheP[CH(2)]Phe and hPheP[CH(2)]Tyr (K(i) values 66 nM and 67 nM, respectively, for the mixture of four diastereomers). The binding affinities of these inhibitors toward the enzyme are the highest, if considering all compounds containing a phosphorus atom that mimic the transition state of the reaction catalyzed by LAP. To evaluate selectivity of the designed LAP inhibitors, additional tests toward aminopeptidase N (APN) were performed. The key feature, which determines their selectivity, is structure at the P1' position. Aromatic and aliphatic substituents placed at this position strongly interact with the LAP S1' binding pocket, while a significant increase in binding affinity toward APN was observed for compounds containing aromatic versus leucine side chains at the P1' position. The most selective inhibitor, hPheP[CH(2)]Leu, binds to LAP with 15 times higher affinity than to APN. One of the studied compounds, hPheP[CH(2)]Tyr, appeared to be very potent inhibitor of APN (K(i) = 36 nM for the mixture of four diastereomers). The most promising LAP inhibitors designed by computer-aided approach, the phosphonamidate dipeptide analogues, were unstable at pH below 12, because of the P-N bond decomposition, which excluded the possibility of determination of their binding affinities toward LAP.     

Buccal Absorption. III. Simultaneous Diffusion and Metabolism of an Aminopeptidase Substrate in the Hamster Cheek Pouch

The simultaneous diffusion and metabolism of the D- and L-isomers of the aminopeptidase substrate, leucine-p-nitroanilide (LPNA), were examined in vitro in the hamster cheek pouch. L-LPNA was completely hydrolyzed during diffusion across the cheek pouch, whereas D-LPNA crossed the cheek pouch intact. The metabolic barrier appeared to be localized in the epithelium of the cheek pouch. Addition of an aminopeptidase inhibitor, bestatin, to both diffusion cell reservoirs resulted in decreased hydrolysis of L-LPNA. The experimental results were analyzed with a mathematical model which was developed to describe the simultaneous diffusion and metabolism processes. Using this model it was estimated that the rate of diffusion of L-LPNA across the cheek pouch was less than the capacity of the tissue to hydrolyze L-LPNA.     

N-terminal degradation of low molecular weight opioid peptides in human cerebrospinal fluid

Opioid peptides are present in human cerebrospinal fluid (CSF), and their levels are reported to change in some pathologic conditions. However, less is known about their degradation in CSF. In the present study, human CSF was found to contain aminopeptidase activity which hydrolyzed alanyl-, leucyl- and arginyl-naphthylamides in a ratio of 100:28:27. Twelve CSF samples hydrolyzed alanyl-2-naphthylamide and degraded Met5-enkephalin (N-terminal hydrolysis) at rates of 188 +/- 38 and 420 +/- 79 pmol/min/mL respectively. Further, the distribution of alanyl-naphthylamidase activity in individual samples (39-437 pmol/min/mL) was closely correlated with that of Met5-enkephalin degradation (37-833 pmol/min/mL). Both alanyl-naphthylamidase and enkephalin degradation were optimal at pH 7.0 to 7.5 and were inhibited by aminopeptidase inhibitors amastatin (IC50 = 20 nM), bestatin (4-7 microM) and puromycin (30-35 microM). Conversely, degradation was unaffected by inhibitors of neutral endopeptidase (phosphoramidon), carboxypeptidase N (MERGETPA) or angiotensin converting enzyme (captopril). The Km of Met5-enkephalin for the CSF aminopeptidase activity was 201 +/- 19 microM (N = 4). Rates of hydrolysis of the Tyr1-Gly2 bond of larger opioid peptides decreased with increasing peptide length. Pooled, concentrated CSF hydrolyzed Leu5-enkephalin, dynorphin A fragments [1-7], [1-10] and [1-13] and dynorphin A at rates of 2.05 +/- 0.27, 1.27 +/- 0.18, 0.94 +/- 0.06, 0.55 +/- 0.14 and 0.16 +/- 0.03 nmol/min/mL respectively. When analyzed by rocket-immunoelectrophoresis against antisera to aminopeptidase M (EC 3.4.11.2), the concentrated CSF formed an immunoprecipitate which could be stained histochemically for alanyl-naphthylamidase activity. These data are consistent with a significant role for aminopeptidase M activity in the degradation of low molecular weight opioid peptides in human CSF.     

Synthesis of p-hydroxyubenimex

p-Hydroxyubenimex, (2S,3R)-3-amino-2-hydroxy-4-p-hydroxyphenylbutyryl-L-leucine, was synthesized starting from D-tyrosine. The structure and stereochemistry of the synthesized product were confirmed by comparison with p-hydroxyubenimex that was chemically transformed from ubenimex, an aminopeptidase inhibitor of microbial origin. Compared to ubenimex, p-hydroxyubenimex is more active against aminopeptidase B but less active against leucine aminopeptidase. By using the synthetic p-hydroxyubenimex as a reference sample, one of the metabolites of ubenimex was identified as p-hydroxyubenimex. The (2R,3R)-stereoisomer of p-hydroxyubenimex was also prepared. However, its activity against aminopeptidases was much weaker.