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.     

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.     

Study on a prolyl endopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

A prolyl endopeptidase (PEP) was purified to homogeneity from the skeletal muscle of common carp using a procedure involving ammonium sulfate fractionation and column chromatography involving DEAE-Sephacel, Phenyl-Sepharose, DEAE-Sepharose Fast Flow, and hydroxyapatite. The molecular weight of the PEP was 82 kDa as determined by SDS-PAGE. Using Suc-Gly-Pro-MCA as a substrate, the optimal pH and temperature of the purified enzyme were pH 6.0 and 35 °C, respectively, and the K_m and k_cat were 8.33 μM and 1.71 S^?1, respectively. The activity of the PEP was inhibited by SUAM-14746, a specific inhibitor of prolyl endopeptidases, and was partially inhibited by the serine proteinase inhibitors PMSF and Pefabloc SC. According to peptide mass fingerprinting, 12 peptide fragments with a total of 134 amino acid residues were obtained, which were highly identical to prolyl endopeptidases from zebrafish (Danio rerio) and sponge (Amphimedon queenslandica), confirming the purified enzyme was a prolyl endopeptidase. Our present study for the first time reported the existence of a prolyl endopeptidase in fish muscle.     

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 prolyl endopeptidase from pig brain

The prolyl endopeptidase from pig brain was purified to homogeneity according to SDS-gel electrophoresis and visualization with the silver staining procedure. The molecular weight of prolyl endopeptidase was estimated as 70 kDa, and the isoelectric point as 4.9. The molecular properties of prolyl endopeptidase from pig brain are therefore similar to those of prolyl endopeptidases from other mammalian tissues. Diisopropylfluorophosphate, diethylpyrocarbonate and p-chloromercuribenzoic acid are strong irreversible inhibitors of prolyl endopeptidase from pig brain. We showed that diisopropylfluorophosphate und diethylpyrocarbonate act as competitive inhibitors with respect to substrate. Therefore it is assumed that at least one serine and one histidine residue are located at the active site of this enzyme. This result supports the assumption that the prolyl endopeptidase from pig brain is a typical serine protease. Substance P, thyreoliberin, beta-casomorphin-5 and morphiceptin are hydrolysed by prolyl endopeptidase in vitro.     

Characterization of a prolyl endopeptidase (kininase) from human urine using fluorogenic quenched substrates

A prolyl endopeptidase (PE) was purified 83 times from human urine by DEAE-cellulose and Sepharose Mercurial chromatographies. In this work we studied the specificity of PE using different fluorogenics substrates. Further characterization of the enzyme was carried out using BK and it's analogue, Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp, for measure of enzymatic activity of prolyl endopeptidase (Abz=ortho-aminobenzoic acid; EDDnp=N-[2, 4-dinitrophenyl]ethylenediamine). The substrate Abz-FPQ-EDDnp was considered as specific for PE. The endopeptidase PE, with a molecular weight of 45 kDa, was inhibited 100% by EDTA and pOHMB and resistant to PMSF, thyorphan, E64 and phosphoramidon, when we used the mentioned substrates. These results suggest that PE is a metallo endopeptidase that contains a thiol group important for it's activity. It was also able to hydrolyze in Abz-RPPGFSPFRQ-EDDnp the F-R peptide bound, differing from those obtained upon BK molecule, where the enzyme prefer the peptide bound located after double proline. In the substrate Abz-FPQ-EDDnp PE hydrolyzes the P-Q peptide bound. Furthermore the urinary PE is particularly unable to hydrolyze peptides with single prolines such as substance P, neurotensin and LHRH. The determined K(m) for Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp were 0.74 and 0.65 uM, respectively. The optimum pH for the PE activity, using the substrate Abz-RPPGFSPFRQ-EDDnp was approximately 9.0, but using the specific substrate Abz-FPQ-EDDnp was 6.5 and 8.0. Endopeptidases, which are situated at brush border surface from proximal tubules, have an important role in kidney handling of many peptides, which are filtered by the glomerulus. The prolyl endopeptidase located at distal tubule could have an important physiological function in control of kinin formed in this portion. It's known that all components from kallicrein-kinin system like low molecular weigh kininogen and kallikrein are presents in this portion.     

Porcine muscle prolyl endopeptidase: limited proteolysis of tryptic peptides from hemoglobin beta-chains at prolyl and alanyl bonds

Tryptic peptides from hemoglobin (Hb) beta-chains were used as model substrates for limited proteolysis by prolyl endopeptidase (EC 3.4.21.26) from porcine muscle. From the physicochemical and enzymatic properties of prolyl endopeptidase the conditions for routine digestion were established as follows: the molar ratio of enzyme to substrate was 1 to 100, and the reaction was carried out in sodium phosphate buffer (pH 6.4) at 37 degrees C for 4 h. Under these conditions the peptide bonds on the carboxyl terminal sides of proline and alanine residues in the tryptic peptides from Hb beta-chains (with Mr values of less than 2100) were hydrolyzed by the enzyme with the exception of the amino terminal alanyl bond and aminoacyl alanyl bond. In addition, one of five seryl bonds was cleaved by the enzyme. However, the Hb beta-chain itself, Mr 16,600, and its two CNBr-peptides with Mr 10,200 and Mr 6400, respectively, were not hydrolyzed. Under the same conditions a prolyl bond in oxidized B-chains of insulin, Mr 3400, was partially digested, and an alanyl bond was not hydrolyzed. The data indicate that the prolyl endopeptidase is useful for the limited proteolysis of peptides with relative masses of less than 3000 at both prolyl and alanyl bonds.     

Elicitor-induced prolyl hydroxylase from French bean (Phaseolus vulgaris). Localization, purification and properties.

The enzyme prolyl hydroxylase (proline: 2-oxoglutarate dioxygenase, EC 1.14.11.12), induced in suspension-cultured cells of Phaseolus vulgaris L. (French bean) by treatment with an elicitor preparation from the phytopathogenic fungus Colletotrichum lindemuthianum, has been investigated. The enzyme, which catalyses the hydroxylation of poly-L-proline with the stoichiometric decarboxylation of 2-oxoglutarate, has been shown to be localized mainly in smooth endoplasmic reticulum. After solubilization from microsomal membranes, the hydroxylase was purified by ion-exchange chromatography and affinity chromatography on poly-L-proline-Sepharose 4B. The subunit Mr, as assessed by sodium dodecyl sulphate/poly-acrylamide-gel electrophoresis, was 65 000, the subunit apparently being recovered as a doublet: the subunits associate under non-denaturing conditions to give at least a tetramer. The bean hydroxylase has kinetic properties and cofactor requirements similar to those previously reported for the enzyme from other plants. Elicitor treatment of suspension-cultured bean cells leads to a rapid induction of prolyl hydroxylase activity concomitant with induction of a protein: arabinosyl-transferase and increased levels of an arabinosylated hydroxyproline-rich protein.     

Purification and characterization of an extracellular prolyl endopeptidase from Agaricus bisporus

Prolyl endopeptidase [EC 3.4.21.26] was purified to homogeneity from the culture filtrate of Agaricus bisporus by a procedure that comprised ammonium sulfate fractionation, anion-exchange chromatographies on DEAE-Toyopearl and DEAE-Sephadex, hydroxylapatite chromatography, and high-performance liquid chromatography (HPLC) on a TSKgel G 2000 SW column. The overall activity recovery was 8.6%. The enzyme was most active at or around pH 7.5 and was stable in the range of pH 5-9 when checked with Z-Gly-Pro-beta-naphthylamide as a substrate. The isoelectric point of the enzyme was about 4.8. The enzyme was a monomeric protein of molecular weight 78,000 +/- 2,000 as judged by gel permeation chromatography on Sephadex G-150 and electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gel. The enzyme hydrolyzed Pro-X bonds and at least five subsites (S3, S2, S1, S1', and S2') were found to be involved in enzyme-substrate binding. Among them, S2, S1, and S1' subsites of the enzyme showed high stereospecificity. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), Z-Gly-Pro-CH2Cl, Z-Pro-prolinal, Z-Pro-pyrrolidine, Z-Thiopro-pyrrolidine, Z-Pro-thiazolidine, Z-Thioprothiazolidine, and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenyl-methylsulfonyl fluoride (PMSF), E-64, iodoacetamide, or metal chelators. Although the A. bisporus enzyme showed no immunological cross reaction with anti-bovine prolyl endopeptidase antiserum, the other characteristics were quite similar to those of mammalian and plant enzymes.     

Purification and characterization of a prolyl endopeptidase isolated from Aspergillus oryzae

A new fungal strain that was isolated from our library was identified as an Aspergillus oryzae and noted to produce a novel proly endopeptidase. The enzyme was isolated, purified, and characterized. The molecular mass of the prolyl endopeptidase was estimated to be 60 kDa by using SDS-PAGE. Further biochemical characterization assays revealed that the enzyme attained optimal activity at pH 4.0 with acid pH stability from 3.0 to 5.0. Its optimum temperature was 30 °C and residual activity after 30 min incubation at 55 °C was higher than 80 %. The enzyme was activated and stabilized by Ca²⁺ but inhibited by EDTA (10 mM) and Cu²⁺. The K _m and k _cat values of the purified enzyme for different length substrates were also evaluated, and the results imply that the enzyme from A. oryzae possesses higher affinity for the larger substrates. Furthermore, this paper demonstrates for the first time that a prolyl endopeptidase purified from A. oryzae is able to hydrolyze intact casein.     

Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy

Celiac Sprue is a multi-factorial disease characterized by an inflammatory response to ingested wheat gluten and similar proteins in rye and barley. Proline-rich gluten peptides from wheat, rye, and barley are relatively resistant to gastrointestinal digestion, and therefore persist in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. In this study, we characterize the in vitro gluten detoxifying properties of a therapeutically promising prolyl endopeptidase from Myxococcus xanthus (MX PEP), and describe the development of a prototypical enteric-coated capsule containing a pharmacologically useful dose of this enzyme. A high-cell density fed-batch fermentation process was developed for overproduction of recombinant MX PEP in E. coli, yielding 0.25-0.4 g/L purified protein. A simple, scalable purification and lyophilization procedure was established that yields >95% pure, highly active and stable enzyme as a dry powder. The dry powder was blended with excipients and encapsulated in a hard gelatin capsule. The resulting capsule was enteric coated using Eudragit L30-D55 polymer coat, which provided sufficient resistance to gastric conditions (> 1 h in 0.01 M HCl, pH 2 with pepsin) and rapid release under duodenal conditions (15-30 min release in pH 6.0 in the presence of trypsin and chymotrypsin). In conjunction with pancreatic enzymes, MX PEP breaks down whole gluten into a product mixture that is virtually indistinguishable from that generated by the Flavobacterium meningosepticum (FM) PEP as judged by chromatographic assays. Competitive studies involving selected immunogenic peptides mixed with whole gluten reveal that both PEPs have a wide range of substrate specificity. Our results support further in vitro and in vivo evaluation of the MX PEP capsule as an oral therapeutic agent for Celiac Sprue patients.     

A prolyl endopeptidase from murine macrophages, its assay and specific inactivation

The presence of a prolyl endopeptidase in the soluble fraction of murine peritoneal macrophages is reported. The prolyl endopeptidase is apparently highly specific for cleaving peptides after proline residues. A sensitive new fluorogenic assay substrate matching this specificity, benzyloxycarbonyl-Ala-Ala-Pro beta-methoxynaphthylamide, is described. The enzyme is rapidly inactivated by benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone, one of a class of reagents specific for cysteine proteinases, and by diisopropyl fluorophosphate, an inhibitor of serine proteinases. Culture of macrophages with the addition of low levels of benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone to the media allows the selective inhibition of the cytoplasmic enzyme as measured in lysates at the termination of culture. After exposure to inhibitor, macrophages resynthesize the enzyme over a period of days, a process which is inhibited by cycloheximide. Similar amounts of activity were found in both normal peritoneal macrophages and those elicited by prior injection of thioglycollate media. The enzyme from murine macrophages appears similar to that reported in bronchopulmonary lavage fluid and lung tissue and to those isolated from brain and pituitary tissues.     

Extracellular tyrosinase from Streptomyces sp. KY-453: purification and some enzymatic properties

A strain of Streptomyces isolated from soil was found to produce a large amount of tyrosinase (monophenol, dihydroxy-L-phenylalanine: oxygen oxidoreductase: EC 1.14.18.1) extracellularly. The enzyme was purified from the culture filtrate about 550-fold by a series of column chromatographies on Duolite A-2 and CM-cellulose and gel filtration on Sephadex G-100. The purified enzyme appeared homogeneous as judged by disc gel electrophoresis. The enzyme catalyzed the hydroxylation of monophenols and the oxidation of diphenols and was most active at pH 6.8 with dihydroxy-L-phenylalanine (L-DOPA) as the substrate. It was inhibited by kojic acid, diethyldithiocarbamate, and inhibitors obtained from micro-organisms. The isoelectric point of the enzyme was 9.9, and the molecular weight was estimated to be 36,000 by gel filtration on Sephadex G-100 and 29,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, which suggests that the enzyme is a monomer. Metal analysis by atomic absorption spectroscopy indicated that the enzyme contains nearly 1 gram atom of copper per mol.     

Cadmium‐induced toxicity increases prolyl endopeptidase (PREP) expression in the rat testis

During the differentiation of the male gamete, there is a massive remodeling in the shape and architecture of all the cells of the seminiferous epithelium. The cytoskeleton, as well as many associated proteins with it, plays a pivotal role in this process. The testis is particularly susceptible to environmental pollutant, which can lead to injury and impairment of normal spermatozoa production. Cadmium (Cd) is one of the major chemical environmental toxicants in economically developed countries. Food and cigarettes are the main sources of exposure to this element. Here, the protective role of zinc (Zn) to prevent the testicular toxicity in male adult rats after prenatal and during lactation exposure to Cd has been assessed. Altered testicular histology at the interstitial and germinal levels was found, whereas Zn supply completely corrected Cd toxicity. Moreover, the effects of these metals on the testicular expression and localization of the protease prolyl endopeptidase (PREP) were evaluated. Interestingly, the results showed an increase of PREP messenger RNA and protein. Data were corroborated by immunofluorescence. This study raises the possibility of using PREP as a new fertility marker.     

Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

The maltose-regulated mlr-2 gene from the hyperthermophilic archaeon Pyrococcus furiosus having homology to bacterial and eukaryal prolyl endopeptidase (PEPase) was cloned and overexpressed in Escherichia coli. Extracts from recombinant cells were capable of hydrolyzing the PEPase substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide (ZGPpNA) with a temperature optimum between 85 and 90 degrees C. Denaturing gel electrophoresis of purified PEPase showed that enzyme activity was associated with a 70-kDa protein, which is consistent with that predicted from the mlr-2 sequence. However, an apparent molecular mass of 59 kDa was obtained from gel permeation studies. In addition to ZGPpNA (K(Mapp) of 53 microM), PEPase was capable of hydrolyzing azocasein, although at a low rate. No activity was detected when ZGPpNA was replaced by substrates for carboxypeptidase A and B, chymotrypsin, subtilisin, and neutral endopeptidase. N-[N-(L-3-trans-Carboxirane-2-carbonyl)-L-Leu]-agmatine (E-64) and tosyl-L-Lys chloromethyl ketone did not inhibit PEPase activity. Both phenylmethylsulfonyl fluoride and diprotin A inhibited ZGPpNA cleavage, the latter doing so competitively (K(lapp) of 343 microM). At 100 degrees C, the enzyme displayed some tolerance to sodium dodecyl sulfate treatment. Stability of PEPase over time was dependent on protein concentration; at temperatures above 65 degrees C, dilute samples retained most of their activity after 24 h while the activity of concentrated preparations diminished significantly. This decrease was found to be due, in part, to autoproteolysis. Partially purified PEPase from P. furiosus exhibited the same temperature optimum, molecular weight, and kinetic characteristics as the enzyme overexpressed in E. coli. Extracts from P. furiosus cultures grown in the presence of maltose were approximately sevenfold greater in PEPase activity than those grown without maltose. Activity could not be detected in clarified medium obtained from maltose-grown cultures. We conclude that mlr-2, now called prpA, encodes PEPase; the physiological role of this protease is presently unknown.     

Human lung post-proline endopeptidase: purification and action on vasoactive peptides

Post-proline endopeptidase (PPE, EC 3.4.21.26) was purified 3,450 times from human lung. PPE was routinely assayed with the artificial substrate, carbobenzoxy-glycyl-L-prolyl-p-nitroanilide (Z-Gly-Pro-pNA). The pH optimum was 7.4, and the Mr was 77,000. Thiol blocking agents were strongly inhibitory but serine blocking agents were not inhibitory. No metal ions were required for activity, but heavy metal ions such as Hg2+, Cu2+, Cd2+, and Zn2+ completely inactivated the enzyme. Both dithiothreitol (DTT) and ethylenediaminetetraacetic acid (EDTA) were required to stabilize PPE activity. Michaelis constant values for Z-Gly-Pro-pNA and carbobenzoxy-glycyl-L-prolyl-2-naphthylamide were 0.36 and 0.10 mmol/l, respectively. PPE cleaved vasoactive peptides including bradykinin (BK) and des-(Arg9)-BK (Pro3-Gly4 and Pro7-Phe8 bonds), angiotensins I and II (Pro7-Phe8 bond), substance P (Pro4-Gln5 bond), and oxytocin (Pro7-Leu8 bond). Each of these peptides inhibited PPE-catalyzed hydrolysis of Z-Gly-Pro-pNA competitively. BK had the lowest Ki value (2.35 mumol/l) and oxytocin had the highest Ki value (84.0 mumol/l). PPE was not inhibited by captopril, a potent inhibitor of angiotensin converting enzyme, which also cleaves the Pro7-Phe8 bond of BK.     

Bacillus subtilis aminopeptidase: purification, characterization and some enzymatic properties.

The extracellular aminopeptidase from Bacillus subtilis was purified 300-fold by a simple procedure which gave a high recovery of enzyme. The native enzyme was shown to be a monomer of molecular weight 46,500 and to contain 1 g-atom of Zn²⁺ per mole of protein. Amino acid analyses demonstrated the protein to be rich in acidic residues and Lys, to possess about 3 residues of Met, and to be devoid of Cys. When activated with 5 mm Co(NO₃)₂ for 90 min the activity of the native enzyme was increased; the amount of activation depended on the identity of the substrate. Cobalt activation involved the reversible binding of 1 g-atom of Co²⁺ per mole of protein, without displacing the native Zn²⁺; K_Co was 1.25 mm. Zinc ions competed with Co²⁺ during activation, a process characterized by a _KZn of 28 μm. Ions other than Co²⁺ did not appreciably activate the enzyme.