Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The K_m values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.     

Aminopeptidase N from Streptococcus salivarius subsp. thermophilus NCDO 573: purification and properties

A 96 kDa aminopeptidase was purified from Streptococcus salivarius subsp. thermophilus NCDO 573. The enzyme had similar properties to aminopeptidases isolated from lactococci and lactobacilli and showed a high degree of N -terminal amino acid sequence homology to aminopeptidase N from Lactococcus lactis subsp. cremoris. It catalysed the hydrolysis of a range of aminoacyl 4-nitroanilides and 7-amido-4-methylcoumarin derivatives, dipeptides, tripeptides and oligopeptides. In common with aminopeptidases from other lactic acid bacteria, the enzyme from Strep. salivarius subsp. thermophilus showed highest activity with lysyl derivatives but was also very active with arginyl and leucyl derivatives. Relative activity with alanyl, phenylalanyl, tyrosyl, seryl and valyl derivatives was considerably lower and with glycyl, glutamyl and prolyl derivatives almost negligible. The aminopeptidase also catalysed the hydrolysis of dipeptides and tripeptides but mostly at rates much less than that with L-lysyl-4-nitroanilide and oligopeptides. The enzyme catalysed the successive hydrolysis of various amino acid residues from the N -terminus of several oligopeptides but it was unable to cleave peptide bonds on the N -terminal side of a proline residue.     

Purification and Characterization of a Neuropeptide-Degrading Aminopeptidase from Human Brain

The major aminopeptidase from human post-mortem brain (occipital cortex) was purified to homogeneity (as judged by polyacrylamide gel electrophoresis) by anion-exchange chromatography (two steps) and gel filtration (two steps). The molecular weight of the enzyme was estimated as 105,000 from gel filtration. Maximum activity was obtained in the presence of 0.5 mM Ca2+ and 1 mM 2-mercaptoethanol at pH 7.3. Enzyme activity was lost on freezing and thawing or on lyophilization. The enzyme was inhibited by metal-ion chelating agents, sulphydryl blocking agents, bestatin, and puromycin. A series of amino acyl-7-amido-4-methylcoumarins was hydrolysed by the enzyme, with the alanyl derivative being hydrolysed most rapidly (Km 170 microM). Specificity studies with a series of alanine dipeptides suggested that a hydrophobic second residue favoured hydrolysis. Several naturally occurring neuropeptides, including Leu5-enkephalin (Km 180 microM), cholecystokinin octapeptide, and Arg8-vasopressin, were hydrolysed by the aminopeptidase. In a series of opioid peptides, increasing chain length led to decreased susceptibility to hydrolysis. Sulphation of the Tyr1 residue of Leu5-enkephalin and the Tyr2 residue of cholecystokinin octapeptide made the peptides more resistant to hydrolysis.     

Cloning, expression, and characterization of tomato (Lycopersicon esculentum) aminopeptidase P.

A cDNA (LeAPP2) was cloned from tomato coding for a 654 amino acid protein of 72.7 kDa. The deduced amino acid sequence was >40% identical with that of mammalian aminopeptidase P, a metalloexopeptidase. All amino acids reported to be important for binding of the active site metals and catalytic activity, respectively, were conserved between LeAPP2 and its mammalian homologues. LeAPP2 was expressed in Escherichia coli in N-terminal fusion with glutathione S-transferase and was purified from bacterial extracts. LeAPP2 was verified as an aminopeptidase P, hydrolyzing the amino-terminal Xaa-Pro bonds of bradykinin and substance P. LeAPP2 also exhibited endoproteolytic activity cleaving, albeit at a reduced rate, the internal -Phe-Gly bond of substance P. Apparent K(m) (15.2 +/- 2.4 microm) and K(m)/k(cat) (0.94 +/- 0.11 mm(-1) x s(-1)) values were obtained for H-Lys(Abz)-Pro-Pro-pNA as the substrate. LeAPP2 activity was maximally stimulated by addition of 4 mm MnCl(2) and to some extent also by Mg(2+), Ca(2+), and Co(2+), whereas other divalent metal ions (Cu(2+), Zn(2+)) were inhibitory. Chelating agents and thiol-modifying reagents inhibited the enzyme. The data are consistent with LeAPP2 being a Mn(II)-dependent metalloprotease. This is the first characterization of a plant aminopeptidase P.     

Purification and characterization of human placental aminopeptidase A

Human placental aminopeptidase A (AAP) was purified 3,900-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100, then subjected to trypsin digestion, zinc sulfate fractionation, chromatographies with DE-52, Sephacryl S-300, and hydroxylapatite, affinity chromatography with Bestatin-Sepharose 4B, and finally immunoaffinity chromatography with the antibody against microsomal leucine aminopeptidase (LAP). Aminopeptidase A was completely separated from leucine aminopeptidase by the immunoaffinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 280,000 by gel filtration. The purified enzyme was most active at pH 7.1 with L-aspartyl-beta-naphthylamide (L-Asp-NA) as substrate; the Km value for this substrate was 4.0 mmol/l in the presence of Ca2+. Human placental aminopeptidase A was markedly activated by alkaline earth metals (Ca2+, Sr2+, Ba2+), but strongly inhibited by metal chelating agents such as EDTA and o-phenanthroline. The highest activity was observed with L-glutamyl-beta-naphthylamide, while only minimal hydrolysis was found with some neutral and basic amino acid beta-naphthylamides.     

Purification and characterization of the major aminopeptidase from human skeletal muscle.

The major aminopeptidase from human quadriceps muscle was purified (as judged by polyacrylamide-gel electrophoresis) by anion-exchange chromatography (two steps) and gel filtration (two steps). The enzyme showed maximum activity at pH 7.3, in the presence of 1 mM-2-mercaptoethanol and 0.5 mM-Ca2+ ions; activation of the enzyme occurred in the presence of several other bivalent cations. Inhibition of activity was obtained in the presence of metal-ion-chelating agents and inhibitors of aminopeptidases and thiol proteinases. The molecular weight of the enzyme was 102 000 (by gel filtration). The enzyme hydrolysed several amino acyl-7-amido-4-methylcoumarin derivatives; highest activity was obtained with alanyl-7-amido-4-methylcoumarin. The enzyme also degraded a series of dipeptides, alanine oligopeptides and some naturally occurring peptides. Of particular interest was the high activity of the enzyme towards the enkephalins.     

A Novel Aminopeptidase with Highest Preference for Lysine

Neuropeptides are formed from sedentary precursors to smaller, active peptides by processing enzymes cleaving at paired basic residues. The process generates peptide intermediates with additional Lys or Arg residues at their NH(2) and COOH termini; the N-terminal basic amino acids are later removed by specific aminopeptidases. We report here a novel lysine-specific aminopeptidase (KAP) of ubiquitous distribution. The enzyme was resolved from puromycin-sensitive aminopeptidase (PSA), aminopeptidase B (APB), and neuron-specific aminopeptidase (NAP). It was purified by FPLC after (NH(4))(2)SO(4) precipitation. The purified KAP had a K(m) of 333 microM with a V(max) of 0.7 nmol Lys ssNA/min/mg protein. N-terminal basic amino acids, Lys in particular, were its favorable substrates. KAP was inhibited by chelating agents and by serine protease inhibitors. It was highly sensitive to aminopeptidase inhibitor bestatin, but insensitive to puromycin and amastatin, showing that KAP is distinct from PSA, NAP, and aminopeptidase A (APA). The 62,000-Da enzyme had a pH optimum at 7.5 and NaCl was its strongest activator. However, metals could not restore KAP's activity after it was dialyzed against EGTA. Our data indicated that rat KAP did not resemble any aminopeptidases as well as the microbial lysine aminopeptidases.     

Mode of action towards oligopeptides and proteins of hydrolase H, a high-molecular-weight aminoendopeptidase from rabbit skeletal muscle

The mode of action towards oligopeptides and proteins of hydrolase H purified from rabbit skeletal muscle was studied. The presence of protamine or alpha-N-benzoylarginine p-nitroanilide (an endopeptidase substrate) changed both the Km and V values of the enzyme towards Leu-beta-naphthylamide (an aminopeptidase substrate). This indicates that the binding site for an endopeptidase substrate is different from that for an aminopeptidase substrate. Hydrolase H as an aminopeptidase displayed broad specificity. The enzyme hydrolyzed various dipeptides readily except the dipeptides containing Pro or an amino acid with a hydrophobic beta-branched chain at the NH2 terminus. Pro and Val at the NH2 terminus of tripeptides were also difficult to release, whereas Ile and Val of tetrapeptides were easily released in contrast with those of dipeptides. The longer the peptide chain of Glyn (n = 2, 3, 4), the more susceptible was it to hydrolase H. Hydrolase H behaved as an endopeptidase only towards protamine among the proteins tested. The other proteins, casein, bovine serum albumin, myofibrils, troponin, hemoglobin, sarcoplasmic proteins, and myoglobin were probably attacked only by the aminopeptidase activity of the enzyme.     

An evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro

The degradation of thyroliberin (less than Glu-His-Pro-NH2) to its component amino acids by the soluble fraction of guinea pig brain is catalysed by four enzymes namely a pyroglutamate aminopeptidase, a post-proline cleaving enzyme, a post-proline dipeptidyl aminopeptidase and a proline dipeptidase. 1. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868-fold and a yield of 5.7%. In addition to catalysing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate-7-amido-4-methylcoumarin the pyroglutamate aminopeptidase catalysed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin bombesin, bradykinin-potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolysed. 2. The post-proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298-fold and a yield of 10.6%. The post-proline cleaving enzyme catalysed the hydrolysis of thyroliberin and N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. It did not catalyse the hydrolysis of glycylproline-7-amido-4-methylcoumarin or His-Pro-NH2. 3. The post-proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301-fold and a yield of 8.9%. The post-proline dipeptidyl aminopeptidase catalysed the hydrolysis of His-Pro-NH2 and glycylproline-7-amido-4-methylcoumarin but did not exhibit any post-proline cleaving endopeptidase activity against thyroliberin or N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. 4. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2-iodoacetamide (100% inhibition at an inhibitor concentration of 5 microM), the post-proline cleaving enzyme by bacitracin (IC50 = 42 microM) and the post-proline dipeptidyl aminopeptidase by puromycin (IC50 = 46 microM). Because of their specific inhibitory effects these three reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.     

Purification and characterization of an X-prolyl-dipeptidyl peptidase from Lactobacillus sakei

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55 degrees C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu(2+), Hg(2+), and Zn(2+)). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K(m) s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 microM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 microM, respectively. Among peptides, beta-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.     

绮丽刺毛霉的一种新型甘氨酸氨肽酶的研究

研究了产自于绮丽刺毛霉（Actinomucor elegans）的一种甘氨酸氨肽酶。分子筛层析表明该酶的天然分子的分子量为320kD,SDSPAGE分析表明蛋白质的亚基分子量为565kD。该酶水解含有甘氨酸残基的底物（如glycinenaphthylamine）的效率要较其它氨基酸残基高得多。该酶的最佳反应温度为30℃,最佳pH为8.0。酶的Km和Kcat值分别为0.24mmol/L与1008 s^-1。1.0mmol/L Zn²⁺,Cu²⁺和Cd²⁺可完全抑制该酶的活性。作用于酶巯基的化学物质对酶活性都有抑制作用。根据络合剂反应的实验结果表明该酶是一种含有金属的酶。当与蛋白酶共同作用时该酶除了甘氨酸外还能提高脯氨酸、精氨酸及谷氨酸的水解率。     

Partial purification and characterization of an ovarian tripeptidyl peptidase: a lysosomal exopeptidase that sequentially releases collagen-related (Gly-Pro-X) triplets

The pregnant hog ovary is a rich source of a novel exopeptidase that catalyzes the release, at pH 5.0, of collagen-related (P3-P2-P1) "triplets" such as Gly-Pro-Met, Gly-Pro-Arg, and Gly-Pro-Ala from arylamide derivatives, provided the N termini are unsubstituted. Corresponding derivatives of related (P2-P1) dipeptides (Pro-Met, Pro-Ala) or (P1) amino acids (Met, Arg, Ala) are not attacked. The enzyme was purified 58-fold from a detergent extract of a water-extracted tissue residue. Activity was determined on Gly-Pro-Met-2-naphthylamide at pH 4.5 and 37 degrees C (Km 0.45 mM; Vmax 722 nmoles/min/mg protein). The responsible Mr 55,000 exopeptidase, termed "tripeptidyl peptidase", forms high-Mr aggregates, belongs to the serine catalytic class, and has a lysosomal localization. Gly-Pro-Ala triplets were released sequentially at pH 5.0 from a Mr 14,000 polypeptide, poly(Gly-Pro-Ala-). When this reaction was coupled to that of homologous dipeptidyl peptidase II, the liberated tripeptides were reduced to dipeptides and free amino acids: (Gly-Pro-Ala)n----nGly-Pro-Ala----nGly-Pro + nAla.     

Multiple intracellular peptidases in Neurospora crassa.

Neurospora crassa possesses multiple intracellular peptidases which display overlapping substrate specificities. They were readily detected by an in situ staining procedure for peptidases separated in polyacrylamide gels, within which the auxilliary enzyme, l-amino acid oxidase, was immobilized. Eleven different intracellular peptidases were identified by electrophoretic separation and verified by their individual patterns of substrate specificities. Most peptide substrates tested were hydrolyzed by several different peptidases. The multiple intracellular peptidases may play overlapping roles in several basic cell processes which involve peptidase activity. The amount of peptidase activity for leucylglycine present in crude extracts of cells grown under widely different conditions was relatively constant, suggesting that this enzyme may be constitutive, although alterations in the amounts of individual peptidase isozymes may occur. A single enzyme, designated peptidase II, was partially purified and obtained free from the other peptidase species. Peptidase II was found to be an aminopeptidase with activity toward many peptides of varied composition and size. It was more active with tripeptides than homologous dipeptides and showed strong activity toward methionine-containing peptides. This enzyme, with a molecular weight of about 37,000, was thermolabile at 65 degrees C and was strongly inhibited by p-hydroxymercuribenzoate, Zn(2+), Co(2+), and Mn(2+), but was insensitive to the serine protease inhibitor phenylmethylsulfonyl fluoride. Peptidase II apparently possesses an essential sulfhydryl group and may be a metalloenzyme.     

Purification and characterization of hen oviduct N alpha-acetyltransferase

Hen oviduct N alpha-acetyltransferase was purified to homogeneity by ammonium sulfate fractionation and DEAE-cellulose, Sepharose 6B, hydroxylapatite, and CoA affinity chromatography. The molecular weights of the native N alpha-acetyltransferase and its protein subunit were estimated as 240,000 and 79,000, respectively. The purified enzyme exhibited a narrow pH optimum centered at 7.8. The enzyme was activated by dithiothreitol, cysteine, glutathione, and beta-mercaptoethanol, but inhibited by Fe2+, Mn2+, Zn2+, Ca2+, Mg2+, and all thiol-specific reagents tested. These findings suggest that a thiol group(s) is essential to the enzyme activity. Substrate specificity experiments of the purified enzyme revealed that (i) the minimal length of a peptide chain required for N alpha-acetylation is 10 residues, (ii) the amino acids, Ala, Ser, Met, and Gly, which are predominantly found in the N termini of N alpha-acetylated proteins, are not the sole determinant of N alpha-acetylation for 10 and more residue peptides, and (iii) N alpha-acetyltransferase recognizes a minute difference in the side chain structure at the N termini of ACTH1-18-NH2 and [Gly1]ACTH1-18-NH2, a productive and a nonproductive substrate, respectively.     

Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.     

Arginine and Lysine Aminopeptidase Activities in Chromaffin Granules of Bovine Adrenal Medulla: Relevance to Prohormone Processing

Abstract: Conversion of prohormones and neuropeptide precursors to smaller, biologically active peptides requires specific proteolytic processing at paired basic residues, which generates intermediate peptides with NH₂ and COOH termini extended with Lys or Arg residues. These basic residues are then removed by aminopeptidase and carboxypeptidase activities, respectively. Among the proteases involved in prohormone processing, the basic residue aminopeptidase activity has not been well studied. This report demonstrates arginine and lysine aminopeptidase activities detected with Arg-methylcoumarinamide (Arg-MCA) and Lys-MCA substrates in neurosecretory vesicles of bovine adrenal medulla [chromaffin granules (CG)], which contain endoproteolytic processing enzymes co-localized with [Met]-enkephalin and other neuropeptides. These arginine and lysine aminopeptidase activities showed many similarities and some differences. Both arginine and lysine aminopeptidase activities were stimulated by the reducing agent β-mercaptoethanol (β-ME) and inhibited by p-hydroxymercuribenzoate, suggesting involvement of reduced cysteinyl residues. The arginine aminopeptidase activity was stimulated by NaCl (150 mM), but the lysine aminopeptidase activity was minimally affected. Moreover, characteristic β-ME/NaCl-stimulated Arg-MCA cleaving activity and β-ME-stimulated Lys-MCA cleaving activity were detected only in CG and not in other subcellular fractions; these findings indicate the localization of these particular basic residue aminopeptidase activities to secretory vesicles. The arginine and lysine aminopeptidase activities showed pH optima at 6.7 and 7.0, respectively. K_m(app) values for the arginine and lysine aminopeptidase activities were 104 and 160 µM, respectively. Inhibition by the aminopeptidase inhibitors bestatin, amastatin, and arphamenine was observed for Arg-MCA and Lys-MCA cleaving activities. Inhibition by the metal ion chelators indicated that metalloproteases were involved; Co²⁺ stimulated the arginine aminopeptidase activity but was less effective in stimulating lysine aminopeptidase activity. In addition, the lysine aminopeptidase activity was partially inhibited by Ni²⁺ and Zn²⁺ (1 mM), whereas the arginine aminopeptidase activity was minimally affected. These results demonstrate the presence of related arginine and lysine thiol metalloaminopeptidase activities in CG that may participate in prohormone processing.     

Characterization of Membrane-Bound Aminopeptidases from Rat Brain: Identification of the Enkephalin-Degrading Aminopeptidase

Rat brain aminopeptidase activity was solubilized from membranes by incubation with thiols. This novel procedure resulted in the release of the same two aminopeptidases (MI and MII) previously shown to be solubilized by the nonionic detergent Triton X-100. The solubilized aminopeptidases MI and MII were resolved by ion-exchange chromatography and further purified by hydroxylapatite chromatography. Aminopeptidase MI was shown to hydrolyze only the beta-naphthylamides of arginine and lysine whereas aminopeptidase MII exhibited a broad specificity with respect to amino acid beta-naphthylamides. Only aminopeptidase MII hydrolyzed Leu-enkephalin at a significant rate, indicating that this enzyme can account for the membrane-bound enkephalin aminopeptidase activity. The enkephalin-degrading aminopeptidase is potently inhibited by opioid (alpha-neo-endorphin and dynorphin) as well as nonopioid (substance P, somatostatin, and angiotensin I) peptides in the range of 0.2-2.0 microM. The regional distribution of aminopeptidases MI and MII in rat brain are rather different, with aminopeptidase MII distribution more closely paralleling the distribution of opiate receptors.     

The purification and characterization of a proline dipeptidase from guinea pig brain

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.     

An activated by cobalt alkaline aminopeptidase from Bacillus mycoides

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-beta-naphthylamide as a substrate was at 37 degrees 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 AmpS of Bacillus cereus and AP II of B. thuringensis.     

Purification and characterization of a prolyl aminopeptidase from Debaryomyces hansenii

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45 degrees C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg(2+), which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The K(m) for proline-7-amido-4-methylcoumarin was 40 micro M. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.