Evidence for conversion of N-Tyr-MIF-1 into MIF-1 by a specific brain aminopeptidase

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly·NH₂), an immunoreactive neuropeptide exhibiting saturable high affinity binding in rat brain was found to be converted into MIF-1 (Pro-Leu-Gly·NH₂) by a specific brain aminopeptidase present in rat brain homogenates or cytosol, but with low activity associated with synaptosomal plasma membranes and microsomes. Conversion occurred at a rate of 16 μmol per g w/wt per h and was unaffected by puromycin but inhibited by bestatin (I₅₀, 5 × 10^?5 M). Aminopeptidases purified from cytosolic fractions of rat brain (arylamidase), mouse brain (Mn²⁺-activated aminopeptidase) or porcine kidney (leucine aminopeptidase) were inactive towards N-Tyr-MIF-1 but degraded MIF-1 with release of Leu-Gly·NH₂ as detected by RP-HPLC procedures. Morphiceptin (Tyr-Pro-Phe-Pro·NH₂), a μ opioid agonist, also acted as a substrate for the N-Tyr-MIF-1 converting enzyme with cleavage of the Tyr-Pro bond. These tetrapeptides, but not MIF-1 or its N-blocked analogs, were degraded in vitro by a metalloendopeptidase purified from kidney membranes. Since dipeptide products were not detected for crude extracts, a significant role for brain metalloendopeptidase on turnover can be excluded. Thus the results point to the presence of a specific (X-Pro-degrading) aminopeptidase in brain cytosol as an enzyme responsible for converting N-Tyr-MIF-1 and inactivating morphiceptin.     

Characterization of an aminopeptidase from Pseudozyma hubeiensis 31-B and potential applications

Yeast strain 31-B was isolated from the digestive juices of Nepenthes alata as an aminopeptidase producer and identified as Pseudozyma hubeiensis via morphological testing and comparative 26S ribosomal DNA-D1/D2 gene sequence analysis. Strain 31-B produced aminopeptidase as extracellular peptidase, but proteinase activity was not detected in the culture filtrate. The aminopeptidase from strain 31-B was purified from filtered culture medium by (NH₄)₂SO₄ precipitation and four column chromatography steps: Diethylaminoethyl (DEAE)-Toyopearl 650 M, Butyl-Toyopearl 650 M, hydroxylapatite, and Toyopearl HW-55. Sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded the purified enzyme as a single band with molecular mass 75.3 kDa. The optimum temperature and pH were approximately 40 °C and 8.0, respectively. The purified aminopeptidase preferentially hydrolyzed Leu-p-NA and its activity was inhibited by ethylenediaminetetraacetic acid. The isolated aminopeptidase reduced the bitterness of peptides generated from milk casein using a bacterial proteinase. These results show that the aminopeptidase produced by P. hubeiensis 31-B has potential application as a food additive in the dairy industry.     

An intracellular aminopeptidase from Streptomyces rimosus that prefers basic amino acids

An aminopeptidase from the mycelia of Streptomyces rimosus was isolated in an electrophoretically homogeneous form. It was shown to be a monomeric, acidic protein (pI = 4.4, mol. wt. approx. 83,000), with optimal activity at pH 7.1–7.8 and at 35–41° C. The enzyme was fully inhibited by 0.1 mM EDTA or 1 mM o-phenanthroline; the activity was restored upon addition of 0.05 mM Co²⁺, Zn²⁺, or Ni²⁺. Amastatin, bestatin, and puromycin also inhibited the enzyme. The aminopeptidase hydrolyzed amino-acid-2-naphthylamides and various di- to heptapeptides. The highest catalytic coefficients (23 and 19 μM^–1 s^–1) were obtained with Arg- and Lys-2-naphthylamide, followed by Leu-, Phe- and Met-derivatives with one order of magnitude lower catalytic coefficients. Basic or bulky hydrophobic amino acids at the P₁ and/or P₁′ position of peptide substrates were preferred. Acidic amino acids and proline were not accepted. The affinity of the enzyme increased with the length of peptide. According to these properties, S. rimosus intracellular aminopeptidase is distinct from the extracellular leucine aminopeptidase of the same organism and can be classified as an Arg(Lys)-preferring metalloaminopeptidase. Received: 18 January 1996 / Accepted: 19 March 1996     

Characterisation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 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.     

Microsomal methionine aminopeptidase: properties of the detergent-solubilized enzyme

A methionine aminopeptidase (MAP) found in rat liver microsomes behaves as membrane-bound enzyme. Triton-solubilized MAP when chromatographed on DEAE-cellulose columns was separated from other microsomal arylamidases. The enzyme hydrolyzes N-terminal methionine from methionyl-lysyl-bradykinin (Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) being then characterized as a typical aminopeptidase. It also shows preferential arylamidase activity upon Met-2-naphthylamide. MAP was activated by 2-mercaptoethanol and inhibited by p-hydroxymercuribenzoate. Contrarily to other well characterized aminopeptidases, MAP was not affected by EDTA, puromycin or bestatin. Altogether these data suggest that MAP is a unique microsomal enzyme distinct from other previously described aminopeptidases. It could be involved in the removal of methionine from nascent peptides during protein synthesis.     

Evidence that two zinc fingers in the methionine aminopeptidase from Saccharomyces cerevisiae are important for normal growth

Limited proteolysis of intact yeast methionine aminopeptidase (MAP1) with trypsin releases a 34 kDa fragment whose NH₂-terminal sequence begins at Asp70, immediately following Lys69. These results suggest that yeast MAP may have a two-domain structure consisting of an NH₂-terminal zinc finger domain and a C-terminal catalytic domain. To test this, a mutant MAP lacking residues 2–69 was generated, overexpressed, purified and analyzed. Metal ion analyses indicate that 1 mol of wild-type yeast MAP contains 2 mol of zinc ions and at least 1 mol of cobalt ion, whereas 1 mol of the truncated MAP lacking the putative zinc fingers contains only a trace amount of zinc ions but still contains one mole of cobalt ion. These results suggest that the two zinc ions observed in the native yeast MAP are located at the Cys/His rich region and the cobalt ion is located in the catalytic domain. The k.at and Km values of the purified truncated MAP are similar to those of the wild-type MAP when measured with peptide substrates in vitro and it appears to be as active as the wild-type MAP in vivo. However, the truncated MAP is significantly less effective in rescuing the slow growth phenotype of map mutant than the wild-type MAP. These findings suggest that the zinc fingers are essential for normal MAP function in vivo, even though the in vitro enzyme assays indicate that they are not involved in catalysis. In addition, a series of single mutations were generated by changing the cysteines and the histidines in the zinc finger region to serines and arginines, respectively. Analyses of these point mutations provide further evidence that the cysteines and histidines are important for the growth promotion function of yeast MAP.     

l-leucine aminopeptidase production by filamentous Aspergillus fungi

AIMS: To screen various filamentous fungi belonging to Aspergillus spp. producing leucine and methionine aminopeptidases. METHODS AND RESULTS: Twenty-eight Aspergillus strains representing 14 species within the genus were screened for L-leucine aminopeptidase (LAP) production in two media in shake flask fermentation. Two Aspergillus sojae (NRRL 1988 and NRRL 6271) and one Aspergillus oryzae (NRRL 6270) strains were selected as the best producers for further studies. The peak LAP activities were 2.61, 2.59 and 1.30 IU ml(-1) for the three fungi on days 2, 5 and 4 respectively. In addition to LAP, L-methionine aminopeptidase (MAP) activity was also detected. Apart from submerged fermentation, the highest LAP yields by solid-state fermentation were 11.39, 17.40 and 13.02 IU g(-1) dry matter for the above fungi. The temperature and pH optimum of the enzyme was found to be in the range of 65-75 degrees C at pH 8.0-9.0 for all three fungi. Metal ions, Co(2+) and Fe(2+) in 2 mmol l(-1) concentration apparently enhanced the relative enzyme activity and heat stability. CONCLUSIONS: Two A. sojae (NRRL 1988 and NRRL 6271) and one A. oryzae (NRRL 6270) strains were found to be the best producers of LAP and MAP. The preliminary characterization studies revealed that the enzyme is considerably thermostable and belongs to the class metalloenzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of aspergilli were screened and the ability of the fungal aminopeptidase to release a particular N-terminal amino acid along with its high thermal stability, makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40°C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu²⁺ and Cd²⁺. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or β-mercapto-ethanol, while Zn²⁺ or Co²⁺ restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K_m, 0.55 mM) but that it can hydrolyze this substrate at a high rate (V_max, 30 μmol/min per mg of protein).     

Release pattern of aminopeptidase from Biomphalaria glabrata hemocytes subjected to high-level bacterial challenge

Specimens of Biomphalaria glabrata, NIH albino strain, challenged with 2 μl of heat-killed Bacillus megaterium at a concentration of 9 × 10⁹ bacteria/ml, showed significant elevation of serum aminopeptidase activity level at 2 hr postchallenge when compared with the serum activity levels of the enzyme in snails of the other experimental groups at four time intervals postchallenge. It was found that challenge with a heavy dosage of heat-killed bacteria did not cause a shift in the highest level of enzyme activity from 2 hr postchallenge. Also, the activity of aminopeptidase was higher at 2 hr postchallenge with a greater dosage of heat-killed bacteria.     

Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

A new type of neuron-specific aminopeptidase NAP-2 in rat brain synaptosomes

A novel neutral aminopeptidase (NAP-2) was found exclusively in the rat central nervous system (CNS). It was separated from the ubiquitous puromycin-sensitive aminopeptidase (PSA) and the neuron-specific aminopeptidase (NAP) by an automated FPLC-aminopeptidase analyzer. The activity of the neuronal aminopeptidase enriched in the synaptosomes is different from NAP and PSA in distribution and during brain development. The enzyme was purified 2230-fold to apparent homogeneity from rat brain cytosol with 4% recovery by ammonium sulfate fractionation, followed by column chromatography successively on Phenyl-Sepharose, Q-Sepharose, Sephadex G-200, and Mono Q. The single-chain enzyme with a molecular mass of 110kDa has an optimal pH of 7.0 and a pI of 5.6. It splits beta-naphthylamides of amino acid with aliphatic, polar uncharged, positively charged, and aromatic side chain. Leucyl beta-naphthylamide (Leu betaNA) is the best substrate with the highest hydrolytic coefficiency followed by Met betaNA=Arg betaNA=Lys betaNA>Ala betaNA>Tyr betaNA>Phe betaNA. The cysteine-, metallo-, glyco-aminopeptidase releases the N-terminal Tyr from Leu-enkephalin with a K(m) 82microM and a k(cat) of 1.08s(-1), and Met-enkephalin with a K(m) of 106microM and a k(cat) of 2.6s(-1). The puromycin-sensitive enzyme is most susceptible to amastatin with an IC(50) of 0.05microM. The data indicate that the enzyme is a new type of NAP found in rodent. Its possible function in neuron growth, neurodegeneration, and carcinomas is discussed.     

Aminopeptidase P from bovine lung: solubilization,properties, and potential role in bradykinin degradation

Aminopeptidase P was solubilized from bovine lung by sodium deoxycholate extraction of salt-washed, delipidated lung acetone powders. Hydrolysis of the standard aminopeptidase P substrate, Gly-Pro-Hyp, as well as cleavage of Arg-Pro-Pro and the Arg¹-Pro² bond of bradykinin, co-eluted from a Mono Q anion exchange column and demonstrated identical inhibitory profiles suggesting that all activities were functions of the same enzyme. The metal chelator, 1,10-phenanthroline, completely inhibited activity suggesting that aminopeptidase P is a metallopeptidase. 2-Mercaptoethanol was both a potent and specific inhibitor of the enzyme (at 4 mM). A variety of other peptidase inhibitors showed either no effect or failed to completely inhibit even at high concentrations. The inhibitory profile and substrate specificity differ considerably from previous reports claiming to study the properties of this enzyme. Evidence is provided that aminopeptidase P may have an important role in the pulmonary degradation of the potent vasoactive peptide, bradykinin.Abbreviations X--NA aminoacyl--naphthylamide - DFP diisopropylphosphofluoridate - HPLC high performance liquid chromatography - APP aminopeptidase P - APM aminopeptidase M - DAP IV dipeptidyl-aminopeptidase IV     

Leucine aminopeptidase in exsheathing fluid of North American and Australian Haemonchus contortus

Rogers W. P. and Brooks F. 1978. Leucine aminopeptidase in exsheathing fluid of north American and Australian Haemonchus contortus. International Journal for Parasitology8: 55–58. Juveniles of Haemonchus contortus from north America and Australia produced exsheathing fluid containing leucine aminopeptidase when stimulated in tetraborate-carbon dioxide medium. Exsheathment in this medium was inhibited by 1, 10-phenanthroline, 10^?3M, and this inhibition was largely reversed by Zn²⁺, 10^?3M. This supports the view that the enzyme is produced by the juveniles and that it is concerned in exsheathment.     

Lapstatin, a new aminopeptidase inhibitor produced by Streptomyces rimosus, inhibits autogenous aminopeptidases

Lapstatin, a low-molecular-weight aminopeptidase inhibitor, was purified to homogeneity from Streptomyces rimosus culture filtrates. The purification procedure included extraction with methanol, followed by chromatography on Dowex 50WX4, AG50WX4, and HPLC RP C₁₈ columns. By amino acid analysis, mass spectrometry, and NMR spectroscopy, the structure of lapstatin was shown to be 3-amino-2-hydroxy-4-methylpentanoylvaline. Lapstatin inhibited the extracellular leucine aminopeptidases from Streptomyces rimosus, Streptomyces griseus, and Aeromonas proteolytica with an IC₅₀ in the range of 0.3–2.4 μM. IC₅₀ values for other enzymes tested were at least tenfold higher. Leucine aminopeptidase from Streptomyces griseus was inhibited in a competitive manner, with an inhibition constant of 5 × 10^–7 M. Lapstatin is the first low-molecular-weight compound isolated from streptomycetes shown to inhibit an autogenous aminopeptidase. Received: 7 December 1998 / Accepted: 29 March 1999     

Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa

The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co²⁺ rather than Zn²⁺: the k_cat (s⁻¹) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co²⁺, and Zn²⁺ conditions, respectively. Consistently, addition of low concentrations of Co²⁺ to PaAP previously saturated with Zn²⁺ greatly enhanced the enzymatic activity, suggesting that Co²⁺ may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co²⁺ or Zn²⁺ commonly showed two metal ions in the active site coordinated with three conserved residues and a bicarbonate ion in a tetragonal geometry. However, Co²⁺- and Zn²⁺-bound structures showed no noticeable alterations relevant to differential effects of metal species, except the relative orientation of Glu-265, a general base in the active site. The characterization of mutant PaAP revealed that the first metal binding site is primarily responsible for metal preference. Similar to PaAP, Streptococcus pneumonia glutamyl aminopeptidase (SpGP), belonging to aminopeptidase family M42 in clan MH, also showed requirement for Co²⁺ for maximum activity. These results proposed that clan MH peptidases might be a cocatalytic cobalt peptidase rather than a zinc-dependent peptidase.     

Purification and characterization of tripeptide aminopeptidase from bovine dental follicles

Tripeptide aminopeptidase (EC 3.4.11.4) was purified from bovine dental follicles by a series of chromatographies. Purified enzyme had a specific activity of 59.5 units per mg protein with L-prolyl-glycylglycine as substrate. The pH optimum was 8.0. The purified native enzyme had a Mr of 230,000 and was shown to be a tetramer of subunit Mr of 58,000. The isoelectric point was pH 7.0. The enzyme was inhibited 5-5-dithio-bis (2-nitrobenzoic acid),o-phenanthroline, and bestatin. Substrate specificity studies indicated that the enzyme specifically hydrolyzes the N-terminal amino acid residue from tripeptides only.     

Heterodimeric Aminopeptidase A from Bacillus licheniformis NS115

An aminopeptidase A (EC 3.4.11.7) was purified to homogeneity from Bacillus licheniformis NS115 and its enzymatic properties were characterized. The enzyme had an apparent molecular mass of 64 kDa, consisting of heterodimeric 42 kDa and 22 kDa subunits, and is a new enzyme from N-terminal analysis of heavy and light subunits. The light suhunit had no catalytic activity against the substrate and apparent K_m values of heavy and whole enzyme were 0.26 and 0.087 mM of γ-glutamyl-p-nitroanilide, respectively.     

Purification,crystallization, and preliminary X-ray analysis of PepX,an X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis

The X-prolyl dipeptidyl aminopeptidase PepX, a serine peptidase isolated originally from Lactococcus lactis subsp lactis NCDO 763, was cloned and overproduced in Escherichia coli. The enzyme was isolated in its active form in two purification steps. Crystals of PepX were grown by the hanging drop vapor diffusion method using polyethyleneglycol 4000 as precipitant at pH 5.0. The crystals are orthorhombic with cell dimensions a = 92.8 Å, b = 102.6 Å, and c = 101.6 Å, space group P2₁2₁2, and probably contain one monomer of 87.5 kDa in the asymmetric unit. The crystals, very stable under X-rays, diffract to at least 2.2 Å and are suitable for high-resolution structural analysis. © 1995 Wiley-Liss, Inc.     

Purification and Characterization of Aminopeptidase B from Escherichia coli K-12

Aminopeptidase B, which is one of the four cysteinyl-glycinases 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 aminopeptidase 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. α-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 k_cat/K_m for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.     

Purification and Properties of an Aminopeptidase from a Protamine-degrading Marine Bacterium

A protamine-degrading marine bacterium was isolated from marine soil and identified as Aevomonas salmonicida subsp. based on its taxonomical characteristics. An alanine-specific aminopeptidase, called aminopeptidase K, from an extract of the strain was purified and characterized. The aminopeptidase K was purified about 80-fold by fractionation with ammonium sulfate and column chromatography on QA-52 cellulose, Phenyl Superose and Superose 12. The purified enzyme is composed of 6 subunits of 86 kDa with a molecular mass of 520 kDa according to gel filtration and SDS–PAGE. The N-terminal sequence of the enzyme was H · Gly-Gln-GIn-Pro-Gln-Ile-Lys-Try-Tyr-His-Asp-Tyr-Asp-Ala-Pro-Asp-Tyr-Tyr-Ile-Thr-. It is inhibited by monoiodoacetate, N-ethylmaleimide, and puromycin. The Michaelis constant (K_m) and the maximal rate of hydrolysis (V_max) were, respectively, 0.28 mm and 49.4 μmol/min/mg for the l-Ala-β-naphthylamide substrate. The optimum pH and optimum temperature were 6.5 and 45°C, respectively. The purified enzyme was highly specific to l-Ala-β-naphthylamide.