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.     

Characterization of a peptidase fromDiplococcus pneumoniae

Some properties of a purified peptidase fromDiplococcus pneumoniae have been studied. The enzyme has a broad pH optimum between 6 and 8 and a K_m (onl-leucylglycylglycine) of 2.8mm. It is activated by low levels of Hg⁺⁺ and is inhibited by Mn⁺⁺, Co⁺⁺, β-mercaptoethanol and EDTA. Substrate specificity studies show that the enzyme is an exopeptidase of the aminopeptidase type, most active on tripeptide substrates bearing bulky substituents at the NH₂ ⁻ terminal end.     

A membrane-bound aminopeptidase isolated from monkey brain and its action on enkephalin

A membrane-bound aminopeptidase which cleaves the tyrosin-glycine bond of enkephalin was purified about 1600-fold from monkey brain. This aminopeptidase hydrolyzed Leu-enkephalin with a K_m value of 35 μM and also hydrolyzed basic, neutral and aromatic amino acid β-naphthylamides. An apparently homogeneous enzyme consisted of a single polypeptide chain with a molecular weight of approx. 100 000. The optimum pH was in the neutral region. From the analysis of the reaction products, only aminopeptidase activity was detected. The enzyme was inactivated by metal chelators, but the activity could be restored by the addition of divalent cations, such as Co²⁺, Mg²⁺ and Zn²⁺. Puromycin, bestatin and amastatin, which are aminopeptidase inhibitors derived from microorganism, showed strong competitive inhibition of the enzyme, the most potent being amastatin, with a K_i value of 0.02 μM.     

A continuous culture study of the regulation of extracellular protease production inVibrio SA1

During growth ofVibrio SA1 in a lactate-limited chemostat in the presence of 2mm phenylalanine as an inducer, the rate of production of two proteolytic enzymes, namely an endopeptidase and an aminopeptidase, was dependent upon the dilution rate. An optimum in the rate of synthesis of both proteases was observed at a dilution rate of 0.23 h^-1 and enzyme production only occurred between dilution rates of 0.06 and 0.45 h^-1. Without inducer a low rate of aminopeptidase production was found with an optimum at 0.19 h^-1, but only trace amounts of endopeptidase were detectable in the culture. In the presence of inducer the rate of enzyme production increased with increasing dilution rates over the range 0.06 to 0.23 h^-1 which was explained by an increase in saturation of inducer sites. The progressive decrease in the rate of protease production at higher dilution rates was ascribed to an increasing effect of catabolite repression by the increasing concentration of the growth substrate. It was shown that 5mm cyclic AMP could not relieve catabolite repression caused by glucose or lactate. Repression of protease production also occurred in the presence of higher concentrations (5mm) phenylalanine and other amino acids and by ammonium ions. It is suggested that the energy-status of the cell may play an important role in the regulation of protease synthesis inVibrio SA1.This study was supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Streptomyces rimosus extracellular proteases 3. Isolation and characterization of leucine aminopeptidase

Summary A leucine aminopeptidase was purified to homogeneity fromStreptomyces rimosus culture filtrates, which are waste broth of oxytetracycline bioproduction process. Purification procedure includes ultrafiltration and chromatography on CM-Sephadex, AH-Sepharose and FPLC Mono S column. The enzyme is a monomer with molecular weight of 27,500 Daltons and pI of 7.3, stable in broad pH range and up to 70°C. It is a metallo enzyme dependent on Ca²⁺ ions for its full activity. By its specificity it is a true aminopeptidase active on amino acid amide, arylamide, peptide and ester bonds. The hydrolysing activity shows preference for leucine at the N-terminal position of substrates, also acts on aromatic acids and methionine, but does not release glycine, proline, acidic amino acids orD-amino acid residues.     

An Aminopeptidase from Mouse Brain Cytosol That Cleaves N-Terminal Acidic Amino Acid Residues

An aminopeptidase with specificity directed toward peptides with acidic N-terminal amino acid residues has been isolated from mouse brain cytosol. Purification by ion-exchange chromatography and gel filtration resulted in an enzyme that hydrolyzed aspartyl-phenylala-nine methyl ester at a rate of 13.2 μu,mol/min/mg protein at pH 7.5, an increase in specific activity of 1000-fold over that of brain homogenate. Its apparent molecular weight, determined by gel filtration, is ?450,000. Dipeptides with N-terminal aspartyl residues are cleaved preferentially to glutamic-containing analogs, and a neutral amino acid (or histidine) is necessary in the adjacent position. For pep-tides of the form aspartyl-X, relative activity was 100, 81, 71, 66, 19, or 0, where X was alanine, serine, leucine, phenylalanine, histidine, or proline, respectively. Tripep-tides were more rapidly hydrolyzed than dipeptides; however, activity tended to decline with increasing chain length. The acidic aminopeptidase can account for almost all of the activity of brain cytosol toward the N-terminal aspartyl residue of angiotensin II, aspartyl-phenylalanine methyl ester or aspartyl-alanine, and the N-terminal glu-tamyl residue of adrenocorticotropin(5-10). The enzyme was unaffected by bestatin or amastatin. It was inhibited by o-phenanthroline and EDTA. The latter effect could be reversed completely by Zn²⁺ and partially by Mn²⁺ or Mg²⁺; Co²⁺ and Fe²⁺ had no effect; Ca²⁺ was inhibitory. These properties distinguish the brain acidic aminopeptidase from aminopeptidase A isolated from human serum or pig kidney and the aspartyl aminopeptidase of dog kidney.     

Aminopeptidase-N from the Helicoverpa armigera (Hubner) Brush Border Membrane Vesicles as a Receptor of Bacillus thuringiensis Cry1Ac δ-Endotoxin

Brush border membrane vesicles (BBMVs) were prepared from the 2^nd instar larvae of Helicoverpa armigera. Binding of the activated Cry1Ac of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the Cry1Ac type δ-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn⁺⁺ chelating agents 2,2′-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of δ-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-amino-acid sequence showed 60–77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S. Received: 4 January 2001 / Accepted: 6 February 2001     

Purification and some properties of the extracellular protease ofBacillus pumilus

Supernatant of a culture ofBacillus pumilus D 78 was precipitated with ethanol and chromatographed on DEAE- and CM-cellulose to isolate and purify a neutral protease with fibrinolytic and caseinolytic activity. Analysis by ultracentrifugation and immunoelectrophoresis indicate the homogeneity of the purified enzyme with the sedimentation constant s_20,w equal to 2.3. The fibrinolytic activity had a lower heat stability and was also more sensitive to pH higher than 8.0. The caseinolytic activity was stable over a wide range of pH (4.5 to 11.0). The enzyme binds acid dyes and is inhibited by Cu²⁺, Zn²⁺, Ca²⁺ and Fe³⁺, as well as byL-cysteine and KCN at a concentration of 10mM. Likewise, EDTA andp-chloromercuribenzoate show an inhibitory effect.     

The BapF protein from Pseudomonas aeruginosa is a β-peptidyl aminopeptidase

A gene encoding a so far uncharacterized β-peptidyl aminopeptidase from the opportunistic human pathogen Pseudomonas aeruginosa PAO1 was cloned and actively expressed in the heterologue host Escherichia coli. The gene was identified in the genome sequence by its homology to the S58 family of peptidases. The sequence revealed an open reading frame of 1,101 bp with a deduced amino acid sequence of 366 amino acids. The gene was amplified by PCR, ligated into pET22b(+) and was successfully expressed in E. coli BL21 (DE3). It was shown that the enzyme consists of two polypeptides (α- and β-subunit), which are processed from the precursor. The enzyme is specific for N-terminal β-alanyl dipeptides (β-Ala-Xaa). BapF hydrolyses efficiently β-alanine at the N-terminal position, including H-β³hAla-pNA, H–D-β³hAla-pNA and β-Ala-l-His (l-carnosine). d- and l-alaninamide were also hydrolysed by the enzyme.     

Enzymatic properties of the glycine d-alanine aminopeptidase of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji)

The gdaA gene encoding S12 family glycine–d-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the d-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high d-stereospecificity and efficiently released N-terminal glycine and d-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and d-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and d-alanine aminopeptidase activity detected at a pH range of 6 to 9.     

Lack of Intra-cellular Signalling by Angiotensin IV in IRAP Transfected Cells

A number of studies have suggested that angiotensin IV is able to mediate a range of signalling events through a receptor distinct to the well-characterised angiotensin AT₁ and AT₂ receptors. This receptor was termed the AT₄ receptor, but was subsequently identified to be the transmembrane enzyme, insulin regulated aminopeptidase, IRAP. Using HEK293T cells transfected with IRAP we investigated whether angiotensin IV was able to mediate signalling events via this aminopeptidase. No effect of the angiotensin IV analogue, Nle¹-Ang IV, on intracellular calcium or ERK phosphorylation was observed. In addition, the effect of Nle¹-Ang IV on IRAP internalization was investigated and, in contrast to classical ligand-mediated receptor endocytosis, Nle¹-Ang IV (10⁻⁶ M) extends the half-life of IRAP at the plasma membrane. Our results do not support a direct role for Ang IV signalling via IRAP in this system.     

A putative proline iminopeptidase of Thermotoga maritima is a leucine aminopeptidese with lysine-p-nitroanilide hydrolyzing activity

A putative aminopeptidase P gene (TM0042, Swissport Q9WXP9, GeneBank AAD35136) of Thermotoga maritima was cloned and expressed in Escherichia coli BL21 (RIL). The enzyme was purified by the combination of ion exchange chromatography; Q-Sepharose and Mono-Q column. The purified recombinant T. maritima aminopeptidase P enzyme, gave a homogenous protein band with an apparent molecular weight of 40 kDa in SDS-PAGE analysis. The enzyme was purified 23-fold with the specific activity of 16.5 unit/mg with the final recovery of 22%. The enzyme was thermostable up to 90 °C for 30 min. An optimal activity was observed at 90 °C at pH 7.5. The purified enzyme was stable between pH 6.5 and 8 at 80 °C with the optimum of pH 7.5. Based on the amino acid sequence, the enzyme belongs to M 24B family of metalloenzymes. None of the divalent cations enhance the activity of the enzyme while Pb²⁺, Cu²⁺, Co²⁺, Cd²⁺, and Zn²⁺ were inhibitory to the enzyme activity. Divalent cation of Mg²⁺ showed 100% enzyme activity, to a lesser extent, Ca²⁺ and Mn²⁺ whereas strong inhibition of enzyme activity was observed with Zn²⁺ and Cd²⁺. The enzyme designated as putative aminopeptidase P was very low activity in hydrolyzing proline-p-nitroanilide. Kinetic studies on the purified enzyme confirmed that the enzyme is a leucine aminopeptidase. Enzyme also hydrolyzes lysine-p-nitroanilide with efficiency comparable to that of leucine-p-nitroanilide. This is the first report of leucine aminopeptidase with lysine-p-nitroanilide hydrolyzing activity, which belongs to the M 24B family of metalloenzymes.     

An activated by cobalt alkaline aminopeptidase from <Emphasis Type="Italic">Bacillus mycoides</Emphasis>

An intracellular arginine—specific aminopeptidase synthesized by Bacillus mycoides was purified and characterized. The purification procedure for studied aminopeptidase consisted of ammonium sulphate precipitation and three chromatographic steps: anion exchange chromatography and gel permeation chromatography. A molecular weight of ∼50 kDa was estimated for the aminopeptidase by gel permeation chromatography and SDS-PAGE. The optimal activity of the enzyme on arginyl-β-naphthylamide as a substrate was at 37°C and pH 9.0. The enzyme showed maximum specificity for basic amino acids: such as Arg and Lys but was also able to hydrolyze aromatic amino acids: Trp, Tyr, and Phe. Co²⁺ ions activated the enzyme, while Zn²⁺, Cu²⁺, Hg²⁺ and Mn²⁺ inhibited it. The enzyme is a metalloaminopeptidase whose activity is inhibited by typical metalloaminopeptidase inhibitors: EDTA and 1,10-phenanthroline. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to Amp S of Bacillus cereus and APII of B. thuringensis.     

Further Characterization of Aminopeptidase of Rabbit Skeletal Muscle

Further investigation on characterization was conducted on purified neutral aminopeptidase of 160,000 daltons from rabbit skeletal muscle. The enzyme possesses arylamidase activity. The greater part of leucine-β-naphthylamide hydrolyzing activity of the muscle extract was attributed to the enzyme. The Km value for Ala-Gly-Phe-Ala, the most cleavable substrate tested, was 0.25 mm. Substrate inhibition was observed for Val-Val-Val-Ala and Val-Val-Val. The enzyme was inhibited by puromycin in a non-competitive manner, Ki being 4 × 10^?6 m. The enzyme was also inhibited by insulin and the oxidized B-chain of insulin. The tetrapeptide with N-terminal residue of d configuration, tRNA, pyruvate and α-ketoglutarate had no effect on the enzyme. On the basis of all properties determined so far, this muscle aminopeptidase is concluded to be identical to none of the known aminopeptidases from other tissues.     

Isolation and characterization of endogenous modulators for GABA system

Pig brain extracts from both soluble and membrane fractions were found to contain potent inhibitors for GABA synthesizing enzyme, GAD, referred to as endogenous GAD inhibitors (EGIs) and for the binding of GABA agonist, muscimol, referred to as muscimol binding inhibitors (MBIs). EGIs and MBIs were first purified through gel-filtration Bio-Gel P-2 columns, in which multiple activity peaks were observed. One of them appears to be co-eluted with eitherl-glutamate or GABA. However, others are clearly separated froml-glutamate or GABA. EGIs were found to be low MW (<1,800 dalton), heat and acid-base stable, negatively charged, non hydrophobic substances. MBIs were found to be low MW (<1,800 dalton) neutral or positively charged substances. MBIs had no effect on [³H]flunitrazepam (FNZP) binding, indicating that they are not endogenous benzodiazepine receptor ligands and they may act specifically on GABA binding site.Special issue dedicated to Dr. Frederick E. Samson     

Purification,characterization and antitumor activity of l-asparaginase isolated from Pseudomonas stutzeri MB-405

An l-asparaginase produced by Pseudomonas stutzeri MB-405 was isolated and characterized. After initial ammonium sulfate fractionation, the enzyme was purified by consecutive column chromatography on Sephadex G-100, Ca-hydroxylapatite, and DEAE-Sephadex A-50. The 665.5-fold purified enzyme thus obtained has the specific activity of 732.3 units mg protein^-1 with an overall recovery of 27.2%. The apparent M _r of the enzyme under nondenaturing and denaturing conditions was 34 kDa and 33 kDa respectively, and the isoelectric point was 6.38±0.02. It displayed optimum activity at pH 9.0 and 37°C. The enzyme was very specific for l-asparagine and did not hydrolyze L-glutaminate. The K _m of the l-asparaginase was found to be 1.45×10^-4 m towards l-asparagine and was competitively inhibited by 5-diazo-4-oxo-l-norvaline (DONV) with a K _i of 0.03mm. Metal ions such as Mn²⁺, Zn²⁺, Hg²⁺, Fe³⁺, Ni²⁺, and Cd²⁺ potentially inhibited the enzyme activity. The activity was enhanced in the presence of thiol-protecting reagents such as DTT, 2-ME, and glutathione (reduced), but inhibited by PCMB and iodoacetamide. The tumor inhibition study with Dalton's lymphoma tumor cells in vivo indicated that this enzyme possesses antitumor properties.     

Aminopeptidase Co,a new yeast peptidase

A new aminopeptidase — aminopeptidase Co — has been detected in the yeast $\text{Saccharomyces}\text{cerevisiae}$. The enzyme is only active in the presence of Co²⁺ions. Zn^2+- and Mn²⁺ions are inhibitory. The enzyme activity is also inhibited by chelating agents. Of the p-nitroanilide derivatives tested only those containing basic amino acids are cleaved.     

Characterization of membrane-associated arginine aminopeptidase inStreptococcus sanguis 903

Extracts of cytoplasmic membranes ofStreptococcus sanguis 903 were analyzed for aminopeptidase activity by isoelectric focusing in polyacrylamide gel and enzyme staining with 16 different aminopeptidase substrates. A single aminopeptidase with specificity for aminoterminal arginine was detected. The enzyme was stimulated by dithiothreitol and-mercaptoethanol. Urea, sodium dodecyl sulfate (SDS), andp-chloromercuribenzoate were inhibitory. Metal ions had little or no effect on activity, except that Hg²⁺, Cu²⁺, and Ni²⁺ were inhibitory. The pH optimum for activity was at 7.2. The molecular mass estimated by SDS-polyacrylamide gel electrophoresis was 170 kDa.     

Purification and characterization of a novel l-2-amino-Δ2-thiazoline-4-carboxylic acid hydrolase from Pseudomonas sp. strain ON-4a expressed in E. coli

l-2-Amino-Δ²-thiazoline-4-carboxylic acid hydrolase (ATC hydrolase) was purified and characterized from the crude extract of Escherichia coli, in which the gene for ATC hydrolase of Pseudomonas sp. strain ON-4a was expressed. The results of SDS–polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 suggested that the ATC hydrolase was a tetrameric enzyme consisted of identical 25-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 7.0 and 30–35°C, respectively. The enzyme did not require divalent cations for the expression of the activity, and Cu²⁺ and Mn²⁺ ions strongly inhibited the enzyme activity. An inhibition experiment by diethylpyrocarbonic acid, 2-hydroxy-5-nitrobenzyl bromide, and N-bromosuccinimide suggested that tryptophan, cysteine, or/and histidine residues may be involved in the catalytic site of this enzyme. The enzyme was strictly specific for the l-form of d,l-ATC and exhibited high activity for the hydrolysis of l-ATC with the values of K _m (0.35 mM) and V _max (69.0 U/mg protein). This enzyme could not cleave the ring structure of derivatives of thiazole, thiazoline, and thiazolidine tested, except for d,l- and l-ATC. These results show that the ATC hydrolase is a novel enzyme cleaving the carbon–sulfur bond in a ring structure of l-ATC to produce N-carbamoyl-l-cysteine.     

The Binding Specificity of Amino Acid Transport System y+L in Human Erythrocytes is Altered by Monovalent Cations

System y⁺L is a broad-scope amino acid transporter which binds and translocates cationic and neutral amino acids. Na⁺ replacement with K⁺ does not affect lysine transport, but markedly decreases the affinity of the transporter for l-leucine and l-glutamine. This observation suggests that the specificity of system y⁺L varies depending on the ionic composition of the medium. Here we have studied the interaction of the carrier with various amino acids in the presence of Na⁺, K⁺, Li⁺ and guanidinium ion. In agreement with the prediction, the specificity of system y⁺L was altered by the monovalent cations. In the presence of Na⁺, l-leucine was the neutral amino acid that interacted more powerfully. Elongation of the side chain (glycine - l-norleucine) strengthened binding. In contrast, bulkiness at the level of the β carbon was detrimental. In K⁺, the carrier behaved as a cationic amino acid specific carrier, interacting weakly with neutral amino acids. Li⁺ was found to potentiate neutral amino acid binding and in general the apparent affinities were higher than in Na⁺; elongation of the nonpolar side chain made a more important contribution to binding and the carrier was more tolerant towards β carbon substitution. Guanidinium stimulated the interaction of the carrier with neutral amino acids, but the effect was restricted to certain analogues (e.g., l-leucine, l-glutamine, l-methionine). Thus, in the presence of guanidinium, the carrier discriminates sharply among different neutral amino acids. The results suggest that the monovalent cations stabilize different carrier conformations. Received: 22 January 1996/Revised: 26 April 1996