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PubMed1976

The differentiation of proximal and distal tubules in the male rat kidney: the appearance of aldolase isozymes, aminopeptidase and alkaline phosphatase during ontogeny.

1. The specific activities of aminopeptidase, alkaline phosphatase and aldolase isozymes were measured in homogenates of kidneys taken at different stages of ontogeny. The cellular localization of these enzymes was studied in cryostat tissue sections using substrate linked assays for aminopeptidase and alkaline phosphatase and the mixed aggregation immuno-cytochemical technique for aldolase isozymes; local enzyme concentrations were estimated photometrically. 2. The presence of both aldolase-A and aldolase-B was demonstrated in all metanephrogenic cells (and at still higher concentrations in collecting tubule cells) of the rat fetus 16 days after conception and in the undifferentiated cells of the neogenetic zone of kidney up to 8 days after birth; no aminopeptidase or alkaline phosphatase could be found in these cells. 3. Measurements made on stained tissue sections show that the shift towards aldolase-B, seen in homogenate analyses, is due to a change in the relative amounts of proximal tubules. No evidence was seen for repression in the synthesis of aldolase-A or aldolase-B monomers in the different kidney cells during ontogeny. 4. Two transitions in the mode of nephron differentiation were observed: one was shortly after birth, the other followed weaning. Before the first transition the concentrations of the enzymes increased to different degrees, such that the enzymes reached concentrations comparable with those as in the cells of adult rats by 2 to 4 days post partum. After the second transition proximal tubule size and specific activity of brush border membrane enzymes increased 3 fold. In contrast, the distal tubules did not increase significantly in size, but their aldolase-A concentration increased 3 fold. 5. Evidence based on enzyme quantification and morphometry in kidney sections is presented to demonstrate that the proximal tubule cells show functional adaptation by two independent mechanisms: specific amplification of gene expression and hypertrophy. In contrast, the distal tubule shows functional adaptation only by specific amplification of gene expression.

Alkaline Phosphatase↗

PubMed1976

Differentiation of epithelial cells in human jejunum: localization and quantification of aminopeptidase, alkaline phosphatase and aldolase isozymes in tissue sections.

Sections from human jejunum were stained histochemically for aminopeptidase and alkaline phosphatase and the aldolase isozymes were detected with the mixed aggregation immuno-cytochemical technique. All enzyme concentrations increased from the bottom to the upper part of the crypt. The concentration of aldolase-A per cell was the same in the upper part of the crypt and the villus, whereas the concentration of the other three enzymes was still higher. Therefore, high amounts of aldolase-B, aminopeptidase and alkaline phosphatase are present in cells highly active in absorption in a fashion similar to that found in the proximal tubule cells of kidney. The relatively undifferentiated cells of the crypts contained both aldolase-A and aldolase-B. Alkaline phosphatase gains its full activity later than aminopeptidase. The synthesis of microvillar membrane enzymes comes to an end earlier than that of the cytosol enzymes.

Alkaline Phosphatase↗

PubMed1989

Aminopeptidase activity in the brains of mice with chronic Toxoplasma gondii infections.

Aminopeptidase activity on beta-naphthylamide (NA) substrates was assayed in brain extracts from normal and Toxoplasma gondii-infected mice at 4 months postinfection. Correlations of levels of aminopeptidase activity, Toxoplasma-specific antibody production, and the number of brain cysts were studied in normal and Toxoplasma-infected mice. The Toxoplasma-specific antibody and the formation of cysts were markedly enhanced in the parasite-infected mice. The highest levels of activity for the NA substrates tested were observed in normal mice. In contrast, the activity levels were significantly lower in T. gondii-infected mice than in the corresponding normal mice. These results suggest an association between chronic toxoplasmosis and aminopeptidase activity in the parasite-infected host brain.

Aminopeptidases↗

PubMed1992

Immunogold labelling is a quantitative method as demonstrated by studies on aminopeptidase N in microvillar membrane vesicles.

Microvillar membrane vesicle preparations with varying content of aminopeptidase N were prepared from enterocytes of the pig small intestine. Postembedding immunogold labelling of aminopeptidase N was performed on these glutaraldehyde/paraformaldehyde-fixed, osmium tetroxide-treated and Epon-embedded microvillar membrane vesicles. The number of gold particles per micrometre microvillar membrane (labelling intensity) was calculated and compared to the corresponding enzymatic activity. A very close relationship was found between labelling intensity and aminopeptidase N activity, demonstrating that postembedding immunogold labelling can be used quantitatively.

Aminopeptidases↗

PubMed1976

[On the differentiation of aminopeptidases in urine by a synthetic tripeptide (author's transl)].

In contrast to findings in healthy people two aminopeptidases with alanine-specifity were found in urine samples of patients after allogenic kidney transplantation. The two aminopeptidases revealed different enzymatic and immunologic properties: one alanine-aminopeptidase (AAP) could be specifically inhibited by a synthetic Thr-Phe-Pro-(tertiary butyl) tripeptide, but was not immunoreactive against an antibody, raised against plasma membranes of human kidney tubule. The tripeptide however was not capable to inhibit the second AAP which exhibited immunoreactive precipitins against the antibody. The appearance of the latter AAP had a close relation to tubular damage during graft rejection episode.

Aminopeptidases↗

PubMed1994

Intracellular aminopeptidases in Streptomyces lividans 66.

We have investigated the aminopeptidase activities present in Streptomyces lividans strains. The majority of these activities proved to be intracellular with multiple active species. Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues. Two other broad spectrum aminopeptidases were found to display homology at both the DNA and protein levels. One showed significant homology to PepN proteins, particularly around the putative zinc-binding residues which are important for catalysis. The second broad spectrum activity was not analyzed in detail but showed a different spectrum of substrate specificity to that of PepN.

Amino Acid Sequence↗

PubMed1985

Biosynthesis and intracellular pool of aminopeptidase N in rabbit enterocytes.

A papain treatment at 15 degrees C and pH 7.3 of a microsomal fraction from rabbit enterocytes quantitatively releases the aminopeptidase N integrated in the plasma membranes without solubilizing the enzyme integrated in the intracellular membranes. Working on A+ rabbits, characterized by the presence on the brush-border hydrolases of glycans corresponding to the human blood group A-determinant structure, it was possible to separate the intracellular aminopeptidase into two major molecular forms with or without these determinants. The molecular form devoid of human blood group A antigenicity corresponds to the only stable intermediate of glycosylation, bearing N-linked high mannose oligosaccharides. This endoglycosidase H-sensitive form is fully active and represents in the steady state about 1% of the total cellular aminopeptidase. It contains a cytoplasmic sequence of about 3000 daltons that has not yet been detected in the mature form. The A antigenicity is acquired simultaneously with processing of high mannose glycans to complex glycans. Pulse chase labeling of jejunum loops with [35S]-methionine showed that the complete processing of the transient form synthesized during 10 min takes 1 hr. During the last 30 min of processing, all the newly transformed molecules are transported to the plasma membrane.

Aminopeptidases↗

PubMed1979

The use of dipeptide-p-nitranilides for the study of aminopeptidase specificity.

The use of dipeptide-p-nitranilides for the study of 2 placental aminopeptidases separated on Sephadex G200 helped in establishing some regular features of their specifities. The high-molecular (320,000 daltons) one prefers Phe in position P'1 to Leu, whereas the lower-molecular aminopeptidase (145,000 daltons) prefers Leu. The high-molecular aminopeptidase splits very slowly the N-terminal Leu when Gly is in adjacent position. Leu-Gly-p-NA is therefore an inhibitor of this AP.

Aminopeptidases↗

PubMed1984

Aminopeptidase and caseinolytic activities of Mycoplasma salivarium.

Aminopeptidase activity was demonstrated in Mycoplasma salivarium (ATCC 23064) cells disrupted by sonic vibrations and lyophilized (crude enzymes), and weak endopeptidase or carboxypeptidase activity was also suggested. The crude enzymes were suspended in 0.1 M borate buffer, pH 8.0, containing 0.5% (w/v) sodium deoxycholate, and then the suspensions were centrifuged at 100,000 g for 2 h. Thus separated, the supernatants were applied to a column of Sephacryl S-300. As a result, aminopeptidase activity was separated from caseinolytic activity, which had already been demonstrated in this organism. The aminopeptidase activity was inhibited by o-phenanthroline and stimulated by Mn2+, and the enzyme exhibited a strong affinity for leucine and arginine. On the other hand, the caseinolytic activity was inhibited considerably by o-phenanthroline and Ni2+ and slightly by diisopropyl fluorophosphate and Co2+. The caseinolytic activity was therefore believed to be due mainly to metalloproteinases and partly to serine proteinases.

Aminopeptidases↗

PubMed1998

Plasmodium chabaudi chabaudi and P. falciparum: inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin.

The major leucine aminopeptidase of the rodent malarial parasite Plasmodium chabaudi chabaudi was partially purified using a combination of high-pressure liquid chromatography on a size-exclusion column and affinity chromatography using the aminopeptidase-specific inhibitor bestatin as the ligand. The purified enzyme showed simple Michaelis-Menten kinetics when the fluorogenic peptide analogue leucyl-7-amino-4-methyl-courmarin served as the substrate, and it was strongly inhibited by both bestatin (Ki = 50.7 +/- 21.0 nM) and nitrobestatin (Ki = 2.51 +/- 0.2 nM) in a competitive manner. These inhibitors were also potent blockers of the growth of P. c. chabaudi and the human parasite P. falciparum in culture, and nitrobestatin was again the more potent. Therefore, the leucine aminopeptidase represents an important target to which novel anti-malarial agents could be directed.

Animals↗

PubMed2005

Characterisation of Aspergillus niger prolyl aminopeptidase.

We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger. Homologous genes are present in the genomes of the Eurotiales A. nidulans, A. fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae. Cell extracts of strains overexpressing the gene under the control of its own promoter showed a fourfold to sixfold increase in prolyl aminopeptidase activity, but no change in phenylalanine or leucine aminopeptidase activity. The overexpressed enzyme was subsequently purified and characterised. The enzyme specifically removes N-terminal proline and hydroxyproline residues from peptides. It is the first enzyme of its kind from a eukaryotic organism that has been characterised.

Amino Acid Sequence↗

PubMed1984

Differentiation of endopeptidases and aminopeptidases by high-performance liquid chromatography of reaction products from chromogenic peptide p-nitroanilines as substrates.

A method for differentiating endopeptidases and aminopeptidases on the basis of substrate specificity is presented. Various synthetic chromogenic substrates, succinyl-(Ala)3-p-nitroaniline, succinyl-(Ala)2-p-nitroaniline, (Ala)3-p-nitroaniline, and (Ala)2-p-nitroaniline, were incubated with various peptidases and the incubation mixtures were directly analyzed by high-performance liquid chromatography to determine the splitting patterns of these substrates by the enzymes. The substrates and hydrolyzed products containing the chromophore were separated on a reverse-phase column under isocratic conditions, and the chromophore was specifically detected in the effluent fractions by absorbance measurement at 314 nm. Endopeptidases, leucine aminopeptidase, and dipeptidyl aminopeptidase showed different patterns of cleavage of the substrates. This simple and rapid high-performance liquid chromatographic procedure is suitable for identifying the above activities in different fractions obtained during separation and purification studies. The same approach was applied to the simultaneous determination of three types of endopeptidase activities in rat tissues based on the ability of the enzymes to hydrolyze different sites in succinyl-(Ala)3-p-nitroaniline.

Aminopeptidases↗

PubMed1983

Modification of the Cl- -activated arginine aminopeptidases from rat liver and human erythrocytes: a comparative study.

The amino acid residues important for the catalytic activity of the Cl- -activated arginine aminopeptidases from human erythrocytes and rat liver were studied using enzyme modification. The general inhibition characteristics were similar with both enzymes. Inactivation with 5,5'-dithiobis-(2-nitrobenzoic acid) revealed one essential SH-group per active enzyme unit in both aminopeptidases. L-Arginyl-L-phenylalanine and N-L-arginyl-2-naphthylamide protected the enzymes against inactivation by DTNB, the former substrate being more effective. The rat liver enzyme was more sensitive to DTNB than the erythrocyte enzyme. Titration with DTNB revealed only fast reacting SH-groups in rat liver APB (mean 7.8). The erythrocyte enzyme, however, revealed SH-groups which reacted fast with low concentrations of DTNB, while high concentrations of DTNB or SDS treatment were needed to reveal all enzyme SH-groups (mean 8.0). The presence of at least one essential imidazole group in the erythrocyte enzyme was indicated by photooxidation in the presence of methylene blue, as previously found with the rat liver enzyme (Mäkinen and Hopsu-Havu, 1967, Enzymologia 32, 333). The pH dependence curves of both enzymes also supported the presence of SH- and imidazole groups at or near the active site. Thus, the functional groups identified were the same for both enzymes. Neither enzyme had essential COOH or arginyl groups and they did not contain any zinc. The absence of Zn suggests that the reaction mechanism recently presented by other authors, based on the presence of Zn in the active center, does not apply to the Cl- -activated arginine aminopeptidases. Accordingly, this enzyme group cannot be classified to metallopeptidases.

Aminopeptidases↗

PubMed1985

Chloride as allosteric effector of yeast aminopeptidase I.

Activation of yeast aminopeptidase I by chloride was studied by kinetic methods. Several effects contributed to overall activity enhancement: At low concentrations of Zn2+ (an essential component of aminopeptidase I) chloride increased the amounts of active enzyme by reducing the cooperativity of metal binding. In addition, substrate turnover was enhanced due to increased kcat and a moderate decrease of Km. At high concentrations of Zn2+ substrate saturation curves were sigmoidal. Under these conditions chloride activated by restoring Michaelis-Menten kinetics of substrate turnover. At the same time, reconstitution of active enzyme from apoprotein and Zn2+ was substantially accelerated and its inactivation due to loss of Zn2+ was retarded. Co2+-Substituted aminopeptidase I, although catalytically active, was much less sensitive to chloride activation. Apparent binding constants for chloride, as estimated from its effects on metal binding and catalysis, respectively, were different. This suggests that two independent activation mechanisms may be operative. Both appear to be mediated by conformational changes of the enzyme protein.

Allosteric Regulation↗

PubMed1992

Rapid purification of the Aeromonas proteolytica aminopeptidase: crystallization and preliminary X-ray data.

The heat-stable aminopeptidase from Aeromonas proteolytica has been purified using two new procedures, with the aim of preparing large single crystals for X-ray analysis. In a first procedure, we tried to avoid any drastic conditions capable of inducing microheterogeneities in the protein sample. The enzyme was purified through two chromatographic steps based on hydrophobic interactions and ion exchange. In a second procedure a heat treatment of the protein to a temperature of 70 degrees C over 5 to 8 h was performed. Both procedures led to an electrophoretically homogeneous and crystallizable aminopeptidase; however, unexpectedly, the crystals obtained through the first procedure contained, in addition to the native aminopeptidase, a cleaved form of the enzyme which has been characterized. Only the native protein was present when the second procedure was used. Large crystals obtained with the native protein form, having an approximate size of 0.4 x 0.4 x 0.6 mm, produced an X-ray diffraction pattern that exhibited the symmetry associated with the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22). The unit cell parameters were a = 109.1 A and c = 97.8 A. Assuming one molecule/asymmetric unit, a value of VM = 2.6 A3/Da and an approximate solvent content of 45% could be estimated. Measurable diffraction intensities were observed at a resolution of 2.5 A.

Aeromonas↗

PubMed1992

Purification and properties of an aminopeptidase from rat-liver cytosol.

An aminopeptidase was purified from the rat-liver cytosolic fraction to apparent electrophoretic homogeneity. The enzyme is a monomeric protein of 95 kDa, having an isoelectric point of 4.9. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cysteine. The enzyme hydrolyzed a broad spectrum of amino acid beta-naphthylamides at a neutral pH. The enzyme also hydrolyzed di-, tri-, and oligopeptides, including physiologically active peptides such as enkephalins and Met-Lys-bradykinin. The enzyme was inhibited by metal-chelating agents, sulfhydryl-reactive reagents, N-P-tosyl-L-phenylalaninechloromethyl ketone, N-P-tosyl-L-lysinechloromethyl ketone, and puromycin but not by protease inhibitors of microbial origin. The enzyme was activated by the addition of Co2+ and sulfhydryl compounds. The aminopeptidase enhanced proteolysis when the enzyme was added to the protease assay system with purified rat-liver cytosolic neutral protease, suggesting the cooperative action of aminopeptidase in the overall process of protein degradation.

Amino Acid Sequence↗

PubMed1980

Leucine aminopeptidase as an echo-enzyme of polymorphonuclear neutrophils.

Intact polymorphonuclear neutrophils were modified chemically by a poorly permeable reagent, diazotized sulfanilic acid, and the changes in the activity of 5'-nucleotidase, alkaline phosphodiesterase, and leucine aminopeptidase were examined. Among three plasma membrane enzymes, 5'-nucleotidase activity was hardly detected in the human neutrophils. The activity of alkaline phosphodiesterase was observed in all the neutrophils examined, but was not inhibited by diazotized sulfanilic acid in the guinea-pig neutrophils. On the other hand, the activity of leucine aminopeptidase was not only found but also inhibited by diazotized sulfanilic acid without the inhibition of lactate dehydrogenase, a cytosol enzyme, in all the neutrophils, suggesting that leucine aminopeptidase is located generally on the plasma membrane as an ecto-enzyme in the neutrophils.

Animals↗

PubMed1976

On the subunit structure of particulate aminopeptidase from pig kidney.

Solubilization of particulate aminopeptidase (EC 3.4.11.2) from pig kidney with Triton X-100 yields an aggregate (mol. wt. approx. 10(6)) that decomposes into "free" aminopeptidase (mol. wt. 280 000) either upon autolysis at pH 5 or after exposure to trypsin. Both procedures yield free enzymes that are identical with respect to electrophoretic mobility, enzymatic activity and zinc content. After dissociation, the enzyme resulting from autolysis yields a single subunit of 140 000 molecular weight while the trypsin-treated enzyme produces three fragments (140 000, 95 000 and 48 000 mol. wt.). As the aggregate is formed by subunits 10 000 daltons heavier than those of the free enzyme, the existence of a hydrophobic portion anchoring the enzyme to the membrane might be postulated. Reactivation experiments carried out on the three purified fragments of urea-denatured aminopeptidase show that the 140 000 molecular weight subunit is the only one able to yield an active enzyme (after spontaneous dimerization). It can be concluded that the smaller fragments are artefacts resulting from trypsin degradation during purification.

Aminopeptidases↗