Proline iminopeptidase from Bacillus coagulans: purification and enzymatic properties

Proline iminopeptidase [EC 3.4.11.5] was purified about 2,700-fold from cell-free extract of Bacillus coagulans by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose, and hydroxyapatite, and gel filtration on Sephadex G-150. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.3 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid including proline, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminus. Pro-D-amino acid bonds were also susceptible to the enzyme. The enzyme was completely inhibited by p-chloromercuribenzoate (PCMB) and partially by proline but not by metal chelators, diisopropylphosphorofluoridate (DFP), or phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by incubation with 2-mercaptoethanol. These results and the chromatographic profile on PCMB-T-Sepharose suggest that the enzyme is a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0, and the molecular weight of the enzyme was estimated to be 40,000 by gel filtration on Sephadex G-100 and 35,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, indicating that the enzyme exists as a monomer.     

Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

A membrane-bound aminopeptidase was purified from rat brain, and its activity was assayed by high-pressure liquid chromatography with Met-enkephalin as the substrate. The enzyme was extracted with 1% Triton X-100 and purified by chromatography, successively on DEAE-Sepharose CL-6B, Bio-Gel HTP, and Sephadex G-200 columns. The overall purification was about 1200-fold, with 25% yield. The purified enzyme showed one band on disc gel electrophoresis and two bands on sodium dodecyl sulfate electrophoresis with molecular weights of 62 000 and 66 000. The aminopeptidase has a pH optimum of 7.0, a Km of 0.28 mM, and a Vmax of 45 mumol (mg of protein)-1 min-1 for Met-enkephalin. It releases tyrosine from Met-enkephalin, but it does not split the byproduct. It does not hydrolyze gamma- or beta-endorphin, or dynorphin, but it does hydrolyze neutral and basic aminoacyl beta-naphthylamides. The enzyme is inhibited by the aminopeptidase inhibitors amastatin, bestatin, and bestatin-Gly. Its properties, such as its subcellular localization, substrate specificity, pH optimum, and molecular weight, distinguish it from leucine aminopeptidase, aminopeptidase A, aminopeptidase B, aminopeptidase M, and the soluble aminopeptidase for enkephalin degradation.     

Purification and properties of an aminopeptidase from Alaska pollack, Theragra chalcogramma, roe

An aminopeptidase was isolated from a soluble fraction of Alaska pollack roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-200. The molecular weight of the enzyme was estimated to be 125,000 and 105,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The pH optimum and temperature optimum were 7.2 and 35 degrees C, respectively. The purified enzyme hydrolyzed various alpha-aminoacyl beta-naphthylamides and cleaved L-Ala-beta-naphthylamide most rapidly. Both a sulfhydryl group and a divalent metal ion are essential for activity; however, the enzyme was inhibited when incubated with divalent metal ions. Puromycin, chelating agents, and thiol reagents were effective inhibitors. The enzyme was also inhibited by L-amino acids, in particular glutamic acid. Thus, the Alaska pollack roe aminopeptidase resembles soluble alanyl aminopeptidase [EC 3.4.11.14].     

Post-proline cleaving enzyme (prolyl endopeptidase) from bovine brain

A post-proline cleaving enzyme [prolyl endopeptidase, EC 3.4.21.26] was purified about 3,700-fold from an extract of bovine brain by a series of column chromatographies on DEAE-Sephadex, hydroxyapatite and PCMB-T-Sepharose, and gel filtration on Sephadex G-200 using N-carbobenzoxy-Gly-Pro-beta-naphthylamide (Z-Gly-Pro-2-NNap), thyrotropin releasing hormone (TRH) and oxytocin as substrates. The purified enzyme appeared homogeneous as judged by disc gel and SDS gel electrophoreses. The enzyme was most active at pH 7.5 and 7.2 with Z-Gly-Pro-2-NNap and TRH, respectively, and hydrolyzed peptide bonds involving Pro-X (X=amino acid, peptide, ester and amide) bonds of synthetic substrates, oxytocin, vasopressin, neurotensin, substance P, tuftsin, bradykinin, and insulin B chain. However, the enzyme was inert toward collagen, gelatin, and casein. The enzyme was completely inactivated by diisopropylphosphorofluoridate (DFP), Z-Gly-Pro-chloromethyl ketone and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenylmethane sulfonylfluoride (PMSF) or metal chelators. Determination of the amino acid composition revealed that the enzyme contained 25 half-cystines. Modification of three cysteine residues of the enzyme by PCMB led to complete inactivation. The isoelectric point of the enzyme was 4.8, and the molecular weight was estimated to be 76,000 by ultracentrifugal analysis and 75,000-74,000 by both gel filtration and sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is present as a monomer. These results indicate that the post-proline cleaving enzyme from bovine brain is very similar to those previously purified from lamb brain and kidney in its enzymatic properties, substrate specificity and physicochemical properties, in sharp contrast with the results obtained by Tate, who reported that the bovine brain prolyl endopeptidase was inert toward oxytocin, vasopressin and bradykinin.     

Prolidase from bovine intestine: purification and characterization

Prolidase [iminodipeptidase, EC 3.4.13.9] was highly purified from the cytosol fraction of bovine small intestine by a series of column chromatographies on DEAE-Toyopearl, Sephadex G-150, PCMB-T-Sepharose and hydroxyapatite. The purified enzyme appeared homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.2 with Gly-Pro as substrate. It was stable between pH 5.5 and 8.5 for 30 min at 30 degrees C and retained half of the activity after 15 min at 40 degrees C. It was completely inactivated by p-chloromercuribenzoate (PCMB) but not inhibited by diisopropylphosphorofluoridate (DFP), phenylmethane sulfonylfluoride (PMSF) and metal chelators. Its amino acid composition was determined. Its molecular weight was estimated to be 116,000 by gel filtration on Sephadex G-150 and 56,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that it is a dimer. It hydrolyzed dipeptides represented as X-Pro (X = amino acid).     

Lyophyllum cinerascens aminopeptidase: purification and enzymatic properties

An aminopeptidase (EC 3.4.11.1) was purified from the extract of Lyophyllum cinerascens by ammonium sulfate fractionation and sequential chromatographies on DEAE-Sephadex, Sephadex G-150, HPLC-phenyl-5PW, and HPLC-DEAE-5PW columns, with an activity recovery of 4.6% using Leu-beta-naphthylamide as a substrate. The enzyme was a tetrameric protein of molecular weight 150,000 and was found to be rich in histidine. It exhibited a pH optimum of 7.2 and stability between pH 5.7 and 7.7. The isoelectric point of the enzyme was 4.6. The enzyme catalyzed the hydrolysis of amino acid beta-naphthylamides, Phe greater than Leu greater than Met greater than Tyr greater than Ala greater than Glu, and the differences of the measured kcat's ranged over 2-3 orders of magnitude while many of the amino acid beta-naphthylamides were not hydrolyzed at all. Other interesting comparisons include two aliphatics, Ala vs Leu, and the aromatics, Tyr vs Phe, which show a 30-fold difference in the kcat/Km values. The enzyme also hydrolyzed Leu-Gly-Gly and the B chain of oxidized insulin to release N-terminal leucine and phenylalanine, respectively. The release of N-terminal Phe from the oxidized B chain is interesting in view of the fact that the penultimate residue is Val, an unfavorable amino acid in the beta-naphthylamide series. The enzyme seems to be a true aminopeptidase, requiring the free amino groups and hydrolyzing dipeptide and oligopeptide from the N-terminal end. The enzyme was resistant to the action of amastatin. Neither sulfhydryl reagents nor serine protease inhibitors affected the enzyme activity; however, the enzyme was inhibited weakly by EDTA and bestatin and strongly by diethyl pyrocarbonate.     

Oxytocinase-like enzyme in an ovarian dysgerminoma: a placenta-specific protein

Aminopeptidase from dysgerminoma was purified and characterized using L-leucine-beta-naphthylamide as substrate. The enzyme was resistant to puromycin, methionine, amastatin, bastatin, and EDTA, and it was heat labile at 60 degrees C. The enzyme showed the same electrophoretic mobility as pregnant-patient serum oxytocinase CAP1 on polyacrylamide gel electrophoresis. Km value against S-benzylcysteine-p-nitroanilide was 4.2 X 10(-4) M. Oxytocin and vasopressin competitively inhibited the enzyme activity. Molecular weight of the enzyme was estimated to be 80,000 by Sephadex G-200 column chromatography. These results suggest that aminopeptidase from dysgerminoma is an oxytocinase-like enzyme, a placenta-specific protein.     

Purification and characterization of aminopeptidase N from human plasma

Human plasma aminopeptidase N (EC 3.4.11.2) was homogeneously purified from outdated bank plasma. Purification procedures included ammonium sulfate fractionation, immunoaffinity chromatography, DEAE-cellulose column chromatography, hydroxyapatite column chromatography and Sephadex G-200 gel filtration. The final recovery of the enzyme was 18% and its specific activity was 71.6 mumol/min/mg protein. SDS-polyacrylamide gel disc electrophoresis and analytical ultracentrifugation showed the homogeneity of the enzyme. Equilibrium ultracentrifugation showed a molecular weight of 210,800. SDS-polyacrylamide gel disc electrophoresis indicated that the enzyme was a dimer consisting of two identical subunits. The isoelectric point of the enzyme was 3.9 at 4 degrees C. The amino acid composition of the enzyme was very similar to those of aminopeptidase N from human kidney, small intestine, and placenta which we have reported previously. Neutral sugar accounted for 11.6%. The Km, Vmax and Kcat values and hydrolytic coefficient (Kcat/Km) of the enzyme with L-alanyl-beta-naphthylamide as substrate were 8.7 X 10(-5) mol/l, 85.9 mumol/min/mg protein, 303/s and 3,483/mmol/l/s, respectively. The enzyme was activated by cobalt ions and markedly inhibited by amastatin. Plasma aminopeptidase N was immunologically indistinguishable from kidney aminopeptidase N.     

Purification and Partial Characterization of an Aminopeptidase from Lactobacillus lactis

A surface-bound aminopeptidase of Lactobacillus lactis cells was solubilized with lysozyme, and the extract was subjected to streptomycin sulfate precipitation, ammonium sulfate fractionation, chromatography on Sephadex G-100 and diethylaminoethyl-Sephadex A-50, and preparative polyacrylamide gel electrophoresis. The purified enzyme was homogeneous in disc electrophoretic analysis and consisted of a single polypeptide chain with a molecular weight of 78,000 to 81,000. The optimal pH and optimal temperature for enzyme activity were 6.2 to 7.2 and 47.5 degrees C, respectively, for l-lysine-4-nitroanilide as the substrate. The enzyme was activated by Co and Zn ions and inhibited by Cu, Hg, and Fe ions and by the metal-complexing reagents ethylenediaminetetraacetic acid, 1,10-phenanthroline, and alpha,alpha'-dipyridyl. Higher concentrations of substrate and hydrolysis products also inhibited the activity of the enzyme. The aminopeptidase had broad substrate specificity and hydrolyzed many amino acid arylamides and many peptides with unsubstituted NH(2)-terminal amino acids.     

Isolation and properties of leucine aminopeptidase from Aspergillus oryzae]

Homogenious leucine aminopeptidase is purified from "oryzine"--mixture of enzymes produced by surface culture of Asperigillus oryzae using treatment with activated characoal, followed by DEAE-cellulose and hydroxylapatite chromatographies, Biogel P-100 gel-filtration and polyacrylamide-gel electrophoresis. The enzyme has pH optimum 9.0 and the molecular weight 37500 as estimated by gil-filtration through Sephadex G-100 (superfine) and SDS-polyacrylamide gel electrophoresis. Leucine aminopeptidase from Asp. oryzae has a broad substrate specificity, therefore, cleaving with the highest rate the peptides carrying N-terminal leucine. The enzyme is completely inhibited with EDTA and beta-mercaptoethanol, and it is a metalloenzyme.     

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 human seminal plasma aminopeptidase

A 50.4-fold purification of aminopeptidase is achieved by alcohol precipitation, DEAE-cellulose, CM-cellulose and finally Sephadex G-200 chromatography. On polyacrylamide gel electrophoresis of the purified enzyme after molecular sieving on Sephadex G-200, only one band was obtained, suggesting that the enzyme preparation was obtained almost homogeneous by three steps of column chromatography. Aminopeptidase showed highest activity at pH 7.0, using a buffer system, of 70 mM Na-phosphate. The enzyme was found to be active at 40 degrees C, even at 60 degrees C (80% activity), suggesting that the human seminal plasma enzyme is fairly thermostable. Amongst the various aminoacyl derivatives evaluated as substrates in the present study, L-alanine beta-naphthylamide hydrochloride was found to have the highest rate of hydrolysis. Ovalbumin showed effective cleavage in comparison to that of other natural substrates. The Km value for the purified seminal plasma aminopeptidase towards L-alanine beta-naphthylamide hydrochloride was 4 x 10(-4) M. Hg+2 showed highest inhibitory effect than other metal ions tested in the present study. Concentration causing 50% inhibition of the enzyme (I50) by Hg2+ was 4.7 x 10(-6) M. Inhibition by EDTA at 1 mM concentration in the incubation system was higher than by EGTA and sodium azide, suggesting that the enzyme contains a metallo group at the active site. A 50% inhibition of the enzyme by EDTA was obtained at 5.11 x 10(-3) M. The Ackerman and Potter plot for EDTA inhibition suggests that EDTA is a reversible inhibitor of seminal plasma aminopeptidase. A single molecular form of aminopeptidase was found to be present in human seminal plasma as shown by polyacrylamide activity gel electrophoresis.     

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.     

创新霉素产生菌吲哚丙酮酸甲基转移酶的研究

在创新霉素产生菌济南游动披线菌的无细胞提取物中检测到吲哚丙酮酸甲基转移酶活性,并进行了分离提取。该酶能利用S-腺苷-L-甲硫氨酸对吲哚丙酮酸进行甲基化,它可能作用于创新霉素中间体的甲基化。经过硫酸铵分部盐析和DEA F一纤维素柱层析,得到了纯化60倍的甲基转移酶,比活0．66mu／ms。酶的最适底物是吲哚丙酮酸,最适pH7．5,对于底物s一腺苷一L一甲硫氨酸和吲哚丙酮酸的米氏常数(KⅢ)分别是4×10-,mol／L和1．8×10-7moI／L。用Sephadcx G-150凝胶过滤测得分子量是55000士5000道尔顿。     

Isolation and characterization of cathepsin B from rabbit testis

Cathepsin B (EC 3.4.22.1) has been purified from rabbit testes to apparent homogeneity by chromatography on DE-52, affinity chromatography on organomercurial agarose and subsequent gel filtrations on Sephadex G-75. The enzyme is composed of a single polypeptide of Mr 23,000. Thiol blocking agents and leupeptin abolished the activity of the enzyme completely. The enzyme showed maximum activity at pH 6.0 and 43 degrees C, required 2 mM-cysteine for the optimal activity and had a Km1.45 X 10(-3) M using Z-Arg-beta-naphthylamide as the substrate. However, Z-Arg-Arg-beta-naphthylamide was 12 times more sensitive as a substrate than was Z-Arg-beta-naphthylamide. Rabbit testicular cathepsin B hydrolysed intact proteins. An endogenous inhibitor isolated from the rabbit testes inhibited purified Cathepsin B.     

Purification and characterization of arylamidase from monkey brain.

Arylamidase [EC3.4.11.2] was isolated from monkey brain extract and purified about 2100-fold in approximately 11% yield by a six-step procedure comprising extraction from monkey brain homogenate, ammonium sulfate fractionation, first hydroxylapatite chromatography, DEAE-cellulose chromatography, Sephadex G-200 gell filtration and second hydroxylapatite chromatography. The enzyme showed a single band on polyacrylamide disc electrophoresis and consisted of a single polypeptide chain, as judged by disc electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was strongly inhibited by PCMB, TPCK, and puromycin. Puromycin competitively inhibited the enzyme and the Ii value was about 5 x 10(-7)M. Treatment with EDTA resulted in a loss of enzyme activity. The enzyme activity was restored by addition of Zn2+, Co2+, Mn2+. Among various amino acid beta-naphthylamides, L-alanine beta-naphthylamide was most rapidly hydrolyzed and N-carbobenzoxyl-L-leucine beta-naphthylamide was not hydrolyzed by this enzyme preparation. The molecular weight of the enzyme was 92,000 as determined by gel filtration on Sephadex G-200.     

Partial purification and some properties of gamma-glutamyl transpeptidase from human bile.

1. Gamma-Glutamyl transpepetidase ((5-glutamyl)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2) from human bile has been partially purified using protamine sulphate treatment, DEAE-cellulose chromatography and Sephadex G-200 filtration. The procedure resulted in 150-fold increase in specific acitivity with a 37% yield. 2. The partially purified enzyme showed a single zone of enzyme activity by polyacrylamide gel electrophoresis and eluted in the inner volume of Sephadex G-200. 3. The enzyme had a pH optimum of 8.1 and Km of 1.52 mM using gamma-glutamyl p-nitroanilide as substrate. 4. The effects of cations and different gamma-glutamyl acceptors on the activity of the enzyme are reported. 5. As bile gamma-glutamyl transpeptidase appears to be soluble in the absence of detergents, it is suggested that bile may prove to be a useful source for further studies of the kinetic properties and physiological role of human gamma-glutamyl transpeptidase.     

Immunoaffinity purification and characterization of leucine aminopeptidase from human liver

Leucine aminopeptidase was purified from human liver cytosol to homogeneity, 1538-fold, with a yield of 84.4% by immunoaffinity chromatography. Increases in the activity and the stability of the enzyme were simultaneously observed during the purification procedure, suggesting the presence of some endogenous inhibitor in cytosol. The specific activity and Km value of the enzyme for L-leucine amide were found to be 58.00 mumol/min/mg of protein and 4.02 mM, respectively, at pH 8.0. The molecular weight of the enzyme was determined to be 360,000 by both polyacrylamide gradient gel electrophoresis and Sephadex G-200 gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of native and dimethyl suberimidate cross-linked enzyme indicate that the native enzyme has two subunits of Mr 53,000 (a) and 65,000 (b) and is a hexamer arranged as a trimer of dimers (3 X (a X b)). The optimum pH was 10.5, and the enzyme was stable in the pH range from 7.5-8.5. The enzyme was activated by divalent metal ions, especially by Mg2+ and Mn2+, with no change in Km value. The enzyme was inhibited by metal-chelating agents, indicating it to be a metalloenzyme. Amastatin and bestatin strongly inhibited the enzyme, but leupeptin did not. The enzyme had a broad substrate specificity toward oligopeptides and amino acid amides but had little or no activity toward chromogenic substrates. The enzyme also could hydrolyze natural substrates contained in liver cytosol and accordingly produce many kinds of amino acids commonly found in proteins.     

Purification of hyaluronidase from human placenta.

Hyaluronidase [EC 3.2.1.35] was isolated from human placenta and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150. Its isoelectric point was at pH 5.2 and the molecular weight was 7 X 10(4) based on Sephadex G-200 gel filtration data. This enzyme was very stable at temperatures below 30 degree, but was almost completely inactivated at 60degree within 30 min. Its optimum pH was 3.9, a characteristic property of a lysosomal hyaluronidase. The Michaelis constant was 1.18 x 10(-1) mg per ml with purified hyaluronate. This enzyme depolymerized hyaluronate, chondroitin, chondroitin 4-sulfate and 6-sulfate, and the end product formed from hyaluronate was tetrasaccharide. Its biological diffusing activity was statistically significant on intracutaneous injection of 1.86 mU of the hyaluronidase into the back skine of a rabbit.     

Purification of an Apoplastic β-N-Acetyl-D-Hexosaminidase from Tomato Leaves Infected with Tobacco Mosaic Virus

Systematic infection of tomato (Lycopersicon esculenturn) leaves by tobacco mosaic virus (TMV) increased the levels of β-N-acetyl-D-hexosaminidase activity. The enzyme was purified from intercellular fluid by --20℃ acetone precipitation, CM-Sephadex C-25 ion exchange chromatography, Polybuffer Exchanger 94 chromatofocusing and Sephadex G-150 gel filtration column to homogeneity. The molecular weight obtained by SDS-PAGE and Sephadex G-150 gel filtration was 75 kD and 145 kD respectively. The enzyme hydrolysed p-nitrophenyl-N-acetyl-β-glucosaminide and p-nitrophenyl-N-acetyl-β-galaetosaminide, it was a glycoprotein. Most of the enzyme activity in the TMV-infected tomato leaves was found in the intercellular spaces.