Neuron-specific aminopeptidase and puromycin-sensitive aminopeptidase in rat brain development

Neuron-specific aminopeptidase (NAP) and the ubiquitous puromycin-sensitive aminopeptidase (PSA) were compared in the rat hippocampus during early development. Hippocampus contains the highest amount of NAP determined by a fast-protein liquid chromatography-aminopeptidase analyzer using Leu -naphthylamide as substrate. Both enzymes were found in the hippocampus in all ages. NAP was lower in immature rat; the 19th embryonic-day fetus contained the least. It increased steeply during the prenatal through the early postnatal period, 9-fold by the first month. The rate of increase diminished subsequently, increasing 20% in the second month and 13% in the third. The age-dependent increase in NAP activity was parallel to its protein expression as determined by Western blot. The specific molecular activity (hydrolytic activity/NAP antigenicity) in newborn, 15-day-old, and 30-day-old rats were 1.00, 0.88, and 1.00, respectively. The PSA developmental profile without linear increase in activity was distinct from NAP. PSA activity was higher than NAP in decreasing order, 100–4 times, during the same development span. Similarly, different growth profiles for NAP and PSA were also found in the primary culture of developing cerebellar granule cells. Puromycin (1–5 M) blocked neurite outgrowth and caused apoptosis by nonantibiotic effects. Our data suggest that the synaptosome-enriched NAP plays a role in neuron growth, differentiation, and information programming.     

Soluble and immobilized clostridial aminopeptidase and aminopeptidase P as metal-requiring enzymes

The dependence of enzymatic activity on Co2+ concentration was found to be bell-shaped for the soluble and immobilized clostridial aminopeptidase (alpha-aminoacyl-peptide hydrolase, EC 3.4.11.13) and aminopeptidase P (aminoacylpropyl-peptide hydrolase, EC 3.4.11.9), with maxima in the 3-18 microM range of Co2+ concentration. The Co2+-enzyme association constants derived from the activation of soluble, glass- and cellulose-bound clostridial aminopeptidase by Co2+ were KE-Co = 5.2 X 10(5), 4.5 X 10(6) and 2.0 X 10(5) M-1, respectively; for soluble and glass-bound aminopeptidase P, the KE-Co were 1.5 X 10(5) and 8.2 X 10(5) M-1, respectively. Kinetic measurements indicate the involvement of Co2+ in the enzyme-substrate binding. Cobalt-citrate (Co-cit) acted as a useful metallobuffer and protected both enzymes against inhibition by high concentrations of CoSO4. For association of citrate with Co2+ under the assay conditions, KCo-cit was determined as (5.3 +/- 1.4) X 10(3) M-1 by anodic stripping polarography. In contrast to the rapid association of Co2+ with soluble and glass-bound clostridial aminopeptidase (less than 1 min at 4 degrees C), the dissociation process was very slow (hours to days), being slower for the glass-bound than for the soluble and cellulose-bound enzyme. For aminopeptidase P, both processes were rapid. All the interactions were shown to be reversible.     

The yeast aminopeptidase Y

A metal-dependent aminopeptidase (EC 3.4.11.-), designated APase Y, has been purified to homogeneity by conventional methods. The enzyme is composed of a single polypeptide chain with molecular mass of 102 kilodaltons, estimated by sodium dodecyl sulphate - polyacrylamide gel electrophoresis, with a blocked N-terminal amino acid. It possesses neither endopeptidase nor carboxypeptidase activity and is strongly inhibited by metal-chelating agents, Zn2+, and the protein inhibitor from Neurospora crassa. APase Y is insensitive to Cl anions, S--S reducing reagents, serine protease inhibitors, and the peptidase inhibitor benzamidine. Co2+, Hg2+, and p-chloromercuribenzoate can activate the enzyme up to 22, 20, and 55%, respectively. The holoenzyme is resistant to yeast endopeptidases A, B, and Y, whereas the apoenzyme (obtained after treatment with chelators) is susceptible to the serine endopeptidases B and Y. The enzyme catalyzes hydrolysis of most L peptides possessing free alpha-amino (or imino) group by stepwise removal of N-terminal residue. Peptides with L-leucine at the N terminus are cleaved preferentially. The enzyme is unable to catalyze hydrolysis of X--Pro type peptide bonds, and inefficiently hydrolyzes bonds between Asp--X and Glu--X. L-leucine p-nitroanilide hydrolyzes optimally at pH 8.2 with a Km value of 1 mM. The purified enzyme is stable during storage in 0.05 M phosphate buffer, pH 6.7, containing 40-50% glycerol, at -20 degrees C.     

Structural and immunological evidence for the identity of prolyl aminopeptidase with leucyl aminopeptidase

Prolyl aminopeptidase (EC 3.4.11.5) has been assumed to be a unique enzyme catalyzing specifically the removal of unsubstituted NH2-terminal L-prolyl residues from various peptides and to be distinct from leucyl aminopeptidase (EC 3.4.11.1). In the present study, prolyl aminopeptidases were purified to apparent homogeneity from pig small intestine mucosa and human liver and their NH2-terminal amino acid sequences were determined together with that of pig kidney leucyl aminopeptidase. The NH2-terminal 24-residue sequence of pig intestinal prolyl aminopeptidase was shown to be identical with that of pig kidney leucyl aminopeptidase. The NH2-terminal sequence of human liver prolyl aminopeptidase was also shown to be very similar to that of pig kidney leucyl aminopeptidase. Further, pig intestinal prolyl aminopeptidase and pig kidney leucyl aminopeptidase were immunologically indistinguishable. These lines of evidence strongly suggest that prolyl aminopeptidase is identical with leucyl aminopeptidase.     

Molecular cloning of adipocyte-derived leucine aminopeptidase highly related to placental leucine aminopeptidase/oxytocinase

In the current study, we report the cloning and initial characterization of a novel human cytosolic aminopeptidase named adipocyte-derived leucine aminopeptidase (A-LAP). The sequence encodes a 941-amino acid protein with significant homology (43%) to placental leucine aminopeptidase (P-LAP)/oxytocinase. The predicted A-LAP contains the HEXXH(X)18E consensus sequence, which is characteristic of the M1 family of zinc-metallopeptidases. Although the deduced sequence contains a hydrophobic region near the N-terminus, the enzyme localized mainly in cytoplasm when expressed in COS-7 cells. Northern blot analysis revealed that A-LAP was expressed in all the tissues tested, some of which expressed at least three forms of mRNA, suggesting that the regulation of the gene expression is complex. When aminopeptidase activity of A-LAP was measured with various synthetic substrates, the enzyme revealed a preference for leucine, establishing that A-LAP is a novel leucine aminopeptidase with restricted substrate specificity. The identification of A-LAP, which reveals strong homology to P-LAP, might lead to the definition of a new subfamily of zinc-containing aminopeptidases belonging to the M1 family of metallopeptidases.     

Intramolecularly-quenched fluorescent peptides as fluorogenic substrates ofleucine aminopeptidase and inhibitors of clostridial aminopeptidase.

Fluorogenic oligopeptide derivatives of the type Lys(ABz)-ONBzl, where ABz iso-aminobenzoyl (anthraniloyl), X stands for Ala Phe, or Ala-Ala, and ONBzlis p-nitrobenzyloxy, were synthesized and shown to be hydrolyzed by leucine aminopeptidase. The hydrolysis is accompanied by an increase in fluorescence due to disruptionof the intramolecular quenching of the fluorescent anthraniloyl moiety by the nitrobenzyester group. The spectral characteristics of the compounds are not consistent withan energy transfer mechanism according to F?rster, therefore the quenching isassumed to be caused by a direct encouter between the quenching and the fluorecentgroups. The change in fluorescence that accompanies the enzymic hydrolysis ofthe first peptide bound was used for quantitative measurement of the activity ofthe activity of leucine aminopeptidase and for the determination of some of itskinetic parameters. A bacterial aminopeptidase from Clostrdium histolyticumthat is very similar to leucine aminopeptidase in its substrate specificity inits substrate specificity did not hydrolyze the above peptidederivatives. Thehydrolysis of leucine p-nitroanilide by this enzyme was found to be inhibitedby the three peptides and the corresponding inhibition constants were determined.     

A non-specific aminopeptidase from Aspergillus

A fermentation broth supernatant of the Aspergillus oryzae strain ATCC20386 contains aminopeptidase activity that releases a wide variety of amino acids from natural peptides. The supernatant was fractionated by anion exchange chromatography. Based on the primary amino acid sequence data obtained from proteins in certain fractions, polymerase chain reaction (PCR) primers were made and a PCR product was generated. This PCR product was used to screen an A. oryzae cDNA library from which the full length gene was then obtained. Fusarium venenatum and A. oryzae were used as hosts for gene expression. Transformed strains of both F. venenatum and A. oryzae over-expressed an active aminopeptidase (E.C. 3.4.11), named aminopeptidase II. The recombinant enzyme from both fungal hosts appeared as smears on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After deglycosylation of the N-linked sugars, both samples were a sharp band at approximately 56 kDa and had identical N-terminal amino acid sequences. Aminopeptidase II is a metalloenzyme with, presumably, Zn in the active site. Using various natural peptides and para-nitroanilides (pNAs) of amino acids as substrates, the aminopeptidase was found to be non-specific. Only X-Pro bonds demonstrated resistance to hydrolysis catalyzed by this aminopeptidase. The optimal enzyme activity was observed at pH 9.5 and 55 degrees C. Among amino acid pNAs, Leu-pNA appears to have the highest value of bimolecular constant of 40 min(-1) mM(-1) (k(cat) = 230 min(-1); K(m) = 5.8 mM) at pH 7.5 and 21 degrees C. Among Xaa-Ala-Pro-Tyr-Lys-amide pentapeptides, the velocity of catalytic hydrolysis at pH 7.5 and 21 degrees C was in a decreasing order: Pro, Ala, Leu, Gly and Glu.     

Thermostable aminopeptidase from Pyrococcus horikoshii

From the genome sequence data of the thermophilic archaeon Pyrococcus horikoshii, an open reading frame was found which encodes a protein (332 amino acids) homologous with an endoglucanase from Clostridium thermocellum (42% identity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity). This gene was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined. Although endoglucanase activity was not detected, this protein was found to have aminopeptidase activity to cleave the N-terminal amino acid from a variety of substrates including both N-blocked and non-blocked peptides. The enzyme was stable at 90 degrees C, with the optimum temperature over 90 degrees C. The metal ion bound to this enzyme was calcium, but it was not essential for the aminopeptidase activity. Instead, this enzyme required the cobalt ion for activity. This enzyme is expected to be useful for the removal of N(alpha)-acylated residues in short peptide sequence analysis at high temperatures.     

Distribution and biosynthesis of aminopeptidase N and dipeptidyl aminopeptidase IV in rat small intestine

The regional, cellular and subcellular distribution patterns of aminopeptidase N and dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidase N of brush border membrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidase IV had a more uniform distribution profile with relatively high activity in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cells. Both enzymes were primarily associated with brush border membranes in all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity/mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fold activation of precursor enzyme forms occurs during transfer from the Golgi membranes to the brush border membranes.     

Methionine aminopeptidase 2 and cancer

Methionine aminopeptidase (MetAP) is a bifunctional protein that plays a critical role in the regulation of post-translational processing and protein synthesis. In yeasts and humans, two proteins are known to possess MetAP activity, which are known as MetAP1 and MetAP2. MetAP2 has attracted much more attention than MetAP1 due to the discovery of MetAP2 as a target molecule of the anti-angiogenic compounds, fumallin and ovalicin. MetAP2 plays an important role in the development of different types of cancer. Recently, we observed a high expression of MetAP2 in human colorectal cancer tissues and colon cancer cell lines. In addition, pp60(c-src) expression was correlated with the expression of MetAP2 and N-myristoyltransferase. In this review, we discuss the recent developments of MetAP2 and its inhibitors. Future detailed studies related to MetAP2 and apoptosis will shed light on the involvement of this enzyme in the regulation of various apoptotic factors.     

Endocellular aminopeptidase from Astasia longa

A new aminopeptidase was isolated from the biomass of the flagellate Astasia longa by precipitation with ammonium sulfate, gel filtration, and affinity chromatography on Arginine-Silochrome in 41% yield and with purification degree 490. The enzyme is irreversible inhibited by mercury chloride, EDTA, o-phenanthroline and, partially, bestatin and zinc chloride. It has an optimum pH 8.5 toward the hydrolysis of a synthetic chromogenic substrate Ala-pNA. The enzyme molecular mass is 45 kDa, isoelectric point 5.5, and temperature optimum 45 degrees C. The enzyme most effectively hydrolyzes p-nitroanilides of alanine, arginine, and leucine; it is classified as metalloaminopeptidase.     

Mammalian lens dipeptidyl aminopeptidase III

An intracellular exopeptidase identified as dipeptidyl aminopeptidase III (DAP III) was found to be abundant in the bovine lens. The enzyme contained in aqueous extracts exhibited a marked preference, compared to other dipeptidyl-β-naphthylamides, for the release of Arg-Arg from Arg-Arg-2-NNap at the optimum pH 9.0 and 37°. The K_m for this substrate was estimated to be 2.83 × 10^?5M. Lens DAP III was inhibited by EDTA, p-chloromercuriphenyl sulfonate, and puromycin. Lens aminopeptidase activities measured at pH 7.5 on the β-naphthylamides of leucine, alanine, and arginine, included for comparison, suggested that not only is leucine aminopeptidase abundant, but also other aminopeptidases that appear to include alanine aminopeptidase and aminopeptidase B.     

Aging and cerebral aminopeptidase activity

A comparative study of brain aminopeptidase activity between 18 month old male rats and young adults of 3 months has been carried out utilizing the arylamides Leu-, Arg-, Lys-, Tyr- and Asp-beta-naphthylamide as substrates. Statistical analysis of results showed no significant differences either in areas studied or for enzymatic activities detected when both ages were compared. Two different patterns of regional distribution of enzymatic activity were observed: One came to be as a result of the use of Lys-, Arg-, Leu- or Tyr-beta-naphthylamide and the other as a result of the use of Asp-beta-naphthylamide.     

Properties of Streptomyces griseus aminopeptidase

The paper deals with studying the properties of aminopeptidase isolated from Str. griseus culture fluid. The preparation is characterized by a high specific activity and heat stability, it has no admixtures of carboxypeptidases and proteinases. The enzyme is easily inhibited by EDTA, but the addition of Ca2+ evokes its complete reactivation. A partial recovery of the activity may be also reached under the influence of some other bivalent metals. In hydrolysis of di- and tripeptides it is shown that the enzyme has a preferential effect on the substrates with N-terminal leucine. Peptides with N-terminal alanine, valine and glycine are almost not hydrolyzed. The use of the native insulin and decapeptide with the known amino acidic sequence as substrates shows that aminopeptidase can hydrolyze proteins and peptides with the successive release of some amino acids: phenylalanine, serine triptophane, valine, asparagine, etc. Glycine is difficult for removal and may inhibit the further hydrolysis of the polypeptide chain.