Isolation of an anti-entomopathogenic fungal protein secreted from Pseudomonas aeruginosa BGf-2: An intestinal bacteriam of Blattella germanica (L.).

A bacterial strain (BGf-2) with anti-Beauveria bassiana activity was obtained from the feces of Blattella germanica (L.) and identified as Pseudomonas aeruginosa based on biochemical tests and 16S rRNA sequence analysis. An antifungal protein (A0A0H2ZK06) was purified with Sephadex G-100 column and DEAE-sepharose Fast Flowanion exchange from sterile BGf-2 fermentation liquid. Based on MALDI-TOF MS analysis and protein model building, A0A0H2ZK06 showed homology with Pyrrolidone carboxyl peptidases (pcps). Fermentation liquid and antifungal proteins not only reduced the B. bassiana conidial germination rate but also inhibited hyphal growth. A per os test showed that the mortality of cockroaches decreased after treatment with BGf-2 suspension compared with control. We hypothesized that gut microbes with antifungal activity might play an important role in protect cockroaches from pathogenic fungi.

Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli: purification, biochemical and kinetic characterisation.

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl peptidase type-1 (PAP1). The cloned nucleotide sequence has an ORF coding for a primary sequence of 209 amino acid residues, which displays 98% identity with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary between the two sequences. The recombinant bovine PAP1 with a C-terminal His(6) tag (rBtaPAP1(6H)) was expressed in Escherichia coli XL10-Gold cells and purified by immobilised nickel ion affinity chromatography resulting in a yield of 2.6 mg of PAP1 per litre of culture. Purified rBtaPAP1(6H) had a specific activity of 3633 units mg(-1). SDS-PAGE revealed a band for bovine PAP1 with a molecular weight of approximately 24 kDa, which is in good agreement with previously reported data on PAP1. The K (m) and k (cat) values obtained for rBtaPAP1(6H) were 59 muM and 3.5 s(-1), respectively. The optimum pH for activity was 9.0-9.5 and the optimum temperature was 37 degrees C. rBtaPAP1(6H) was found to have an absolute requirement for the thiol-reducing agent DTT, consistent with the expected property of a cysteine protease. Kinetic studies using the peptides pGlu-His-Pro-NH(2) (TRH), pGlu-Ala and pGlu-Val revealed K (i) values of 44.1, 141 and 652.17 microM, respectively. The lowest K (i), observed for Thyrotropin-releasing Hormone (TRH), indicates that rBtaPAP1(6H) has a higher affinity for tripeptides over dipeptides.

Cloning, expression, and characterization of pyrrolidone carboxyl peptidase from the archaeon Thermococcus litoralis.

The gene encoding pyrrolidone carboxyl peptidase (Pcp) has been cloned from the hyperthermophilic archaeon Thermococcus litoralis. The recombinant enzyme has been expressed in Escherichia coli, purified, and characterized. The T. litoralis Pcp demonstrates strong sequence homology to previously characterized bacterial Pcps. Some investigations have been carried out on enzyme substrate specificity and stability.

Partial purification and some properties of pyroglutamyl peptidase from Enterococcus faecalis.

Pyroglutamyl peptidase was partially purified from Enterococcus faecalis ATCC 19433 by anion-exchange chromatography, gel filtration and salting out after lysis of cell walls with N-acetylmuramidase. Pyroglutamyl peptidase was purified 46-fold with a yield of about 2% based on the total activity of the crude extract. The molecular mass of the bacterial enzyme was estimated to be about 82 kD by gel filtration. The pl of the enzyme was 4.2 and the optimum pH and temperatures for the reaction were 7.2-7.5 and 35-45 degrees C, respectively. The enzyme was relatively stable below 45 degrees C, but almost all the activity was lost after heat-treatment at 55 degrees C for 15 min. The apparent K(m) value for pyroglutamyl-beta-naphthylamide was 0.55 mM. The bacterial enzyme specifically cleaved pyroglutamyl residues from the amino termini of pyroglutamyl compounds, such as Pyr-Asn-Gly, Pyr-His-Gly, Pyr-Ala-Glu, Pyr-Ala, neurotensin, thyrotropin-releasing hormone and bradykinin-potentiator B. However, human IgG and Bence Jones protein, which are high-molecular-mass proteins, were not hydrolysed. Neither derivatives of free amino acids, such as Ala-, Gly-, Pro- and Leu-p-nitroanilide, nor benzoyl-DL-Arg-p-nitroanilide were hydrolysed. The activity was strongly inhibited by thiol-blocking reagents (p-CMB, N-ethylmaleimide, monoiodoacetic acid). In addition, protease inhibitors, such as TLCK and PMSF, reduced the activity by 54 to 73%. These results suggest that the bacterial enzyme is a cysteine protease with sulphydryl residues in its active site and, possibly, histidine or serine residues near the active site.

Bovine brain pyroglutamyl aminopeptidase (type-1): purification and characterisation of a neuropeptide-inactivating peptidase.

Pyroglutamyl aminopeptidase type-1 (PAP-I) is reported to be a soluble, broad specificity aminopeptidase, capable of removing the pyroglutamic acid (pGlu) residue from the amino terminus of pGlu-peptides (e.g. TRH, LHRH, neurotensin and bombesin). The central aim of this study was to undertake, for the first time, the complete purification and characterisation of a PAP activity observed within the cytosolic fraction of bovine whole brain and to compare the properties of the enzyme with previous findings. A series of chromatographic steps (DEAE-Sepharose, Sephacryl S-200 and Activated Thiol Sepharose 4B) generated a soluble PAP activity purified to near homogeneity with a total active yield of 6.6% The enzyme displayed a native molecular mass of approximately 23,700 Da, which compares well with that value obtained under denaturing conditions via SDS-PAGE (24,000 Da), suggesting that the enzyme exists as a monomer. The expression of PAP activity displayed an absolute requirement for the presence of a disulphide bond-reducing agent such as DTT, whilst optimum activity was observed at pH 8.5. strong inhibition of PAP activity was observed with a number of different agents, including transition metal ions, sulphydryl-blocking agents and 2-pyrrolidone (a pGlu analog). A broad pyroglutamyl substrate specificity, which excludes substrates commencing with the pGlu-Pro bond, was also demonstrated for the bovine brain enzyme. Based on a comparison of these findings with those reported for PAP-I in other mammalian tissues, the soluble PAP activity observed in bovine whole brain can tentatively be classified as a pyroglutamyl aminopeptidase type-1 (EC 3.4.19.3).

A study of a synaptosomal thyrotropin releasing hormone-inactivating pyroglutamate aminopeptidase from bovine brain.

Pyroglutamate aminopeptidase type II is a highly specific membrane-bound neuropeptidase that has the ability to remove N-terminal pyroglutamate (Glp) from Thyrotropin Releasing Hormone (Glp-His-Pro-NH2) or very closely related tripeptides or tripeptide amides. In this paper we report on the purification and characterisation of a pyroglutamate aminopeptidase activity from the synaptosomal membranes of bovine brain. The Triton X-100 solubilised enzyme was purified nearly 600-fold by a combination of conventional column chromatography steps with a recovery/yield of 17.0%. Phase-partitioning experiments with Triton X-114 showed the activity to be an integral membrane protein. This detergent-solubilised pyroglutamate aminopeptidase activity was found to have a relative molecular mass of 240 kDa on a calibrated S-200 column. HPLC analysis on a C18 reverse-phase column showed that the purified activity displayed a very narrow substrate specificity cleaving only Thyrotropin Releasing Hormone (TRH) or the very closely related acid-TRH, LHRH (1-3) and the TRH-analogue (methyl-His)-TRH and had a Km of 100 microM for the fluorimetric substrate Glp-His-Pro-methyl-coumarin. The enzyme was inactivated by the metalchelator 1,10-ortho-phenanthroline but showed less sensitivity to EDTA. It also showed some inhibition by thiol protease inhibitors such as iodoacetate and n-ethyl-maleimide. In summary, we have purified a pyroglutamate aminopeptidase from the synaptosomal membrane of bovine brain. This enzyme displays characteristics consistent with it being classified as a PAP type II neuropeptidase with only minor differences from other proteases in this group.

Purification and characterization of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis.

Bacillus subtilis pyrrolidone carboxyl peptidase (Pcp) overexpressed in Escherichia coli was purified to homogeneity in less than 12 h using ammonium sulphate precipitation and hydrophobic interaction chromatography. The enzyme, which removes amino-terminal L-pyroglutamic acid from peptides, appears to be a tetramer of 25,200 molecular mass subunits. The protein cross-reacted with polyclonal antibodies raised against Pcp from Streptococcus pyogenes. The overexpressed enzyme exhibits an absolute substrate specificity towards N-terminal pyroglutamyl residues with a Michaelis constant of 1.04 mM for L-pyroglutamyl-beta-naphthylamide. The enzyme could be used for the removal of pyroglutamyl residues that block amino termini of proteins and peptides before performing Edman sequential degradation.

Pyroglutamate aminopeptidase in rat submaxillary gland.

A significant amount of pyroglutamate aminopeptidase (PGAP) activity was found to be present in 27,000 x g supernatant of rat submaxillary gland, maximum activity being at pH 6.5. EDTA stimulated the enzyme activity by 95% at pH 8.0 while at pH 6.5 it did not have any significant effect. On comparison of its properties submaxillary PGAP appears to be different from brain, pituitary and other reported PGAPs. Submaxillary PGAP could also catalyze efficiently the formation of cyclo (His-Pro) from TRH. Cyclo (His-Pro) formation by submaxillary enzyme was more pronounced than that by liver PGAP.

Molecular characterization of pcp, the structural gene encoding the pyrrolidone carboxylyl peptidase from Streptococcus pyogenes.

This paper describes the cloning of a gene (pcp) coding for pyrrolidone carboxylyl peptidase (PYRase), an enzyme which selectively removes N-terminal pyroglutamic acid residues from polypeptides. This gene was isolated from Streptococcus pyogenes by construction of a gene library with a bacteriophage lambda-derived cosmid-Escherichia coli host system. Nucleotide sequence determination of a 1.3 kb restriction fragment revealed a 645 bp open reading frame encoding a 215-amino-acid product of M(r) 23,135 consistent with the 26 kDa polypeptide obtained from in vivo overexpression in E. coli. Southern hybridization confirmed that pcp is a single-copy gene on the S. pyogenes chromosome. 5' and 3' endpoint mapping of the 0.7 kb specific transcript observed by Northern analysis permitted the identification of transcriptional initiation and termination signals. Structural features of the pcp gene product from S. pyogenes are discussed and compared with that from Bacillus subtilis. The lack of sequence identity with any other known protein or nucleotide sequence suggests that this enzyme belongs to a new class of peptidase.

One step purification and characterization of the pyrrolidone carboxyl peptidase of Streptococcus pyogenes over-expressed in Escherichia coli.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp), an enzyme which selectively removes pyrrolidone carboxylic acid (PCA) from some PCA-peptides and -proteins, was demonstrated in bacteria and in plant, animal and human tissues. In this paper we describe the purification to homogeneity of the enzyme of Streptococcus pyogenes, over-expressed in Escherichia coli. This was achieved, for the first time in one step, by hydrophobic interaction chromatography. Analysis under non-denaturing conditions revealed a molecular mass of 85 kDa and in the presence of sodium dodecyl sulfate gave a molecular mass of 23.5 kDa. Investigations on enzymatic properties showed that the Pcp over-expressed in E. coli disclosed properties similar to those found for the enzyme extracted from S. pyogenes or for some other Pcps studied previously. Thus the over-expressed enzyme should serve as a suitable source for N-terminal unblocking prior to some PCA protein sequencing.

Purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle.

The purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle are described. The characteristics of leucyl aminopeptidase were as follows: optimum activity was at pH 9.5 in the presence of 5 mmol/l Mg2+ or 0.5 mmol Mn2+. No activation of enzyme activity was obtained following addition of other divalent cations or sulphhydryl reagents. Only the leucyl-AMC and methionyl-AMC derivatives were appreciably hydrolysed. The mol mass was estimated as 280 kDa. Approx. 50% inhibition of activity was obtained following addition of p-hydroxymercuriphenyl sulphonate (10 mumol/l), N-ethyl maleimide (2 mmol/l), o-phenanthroline (5 mmol/l), bacitracin (1 mmol/l), amastatin (1 microgram/ml) and bestatin (0.1 mumol/l); no inhibition of activity was obtained in the presence of phenylmethanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). The following oligopeptides were hydrolysed by the enzyme: luliberin 7-10, proctolin and [Leu5]enkephalin; oligopeptides not appreciably hydrolysed included neurotensin, angiotensin-I, substance-P and bradykinin. Pyroglutamyl aminopeptidase had the following characteristics: optimum activity was at pH 8.5 in the presence of 1 mmol/l dithiothreitol (an absolute requirement for maintenance of enzyme activity). Maximum activity was obtained in the absence of divalent cations. Only the pyroglutamyl-AMC derivative was appreciably hydrolysed. The mol mass of this enzyme was estimated as 22 kDa. Approximately 50% inhibition of activity was obtained on addition of phenanthroline (4 mmol/l) and antipain (7 microgram/ml); no inhibition of activity was obtained following addition of phenyl methanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). Only oligopeptides with a pyroglutamyl N-terminal residue (thyroliberin, neurotensin, and luliberin) were hydrolysed by the enzyme.

Rapid inactivation and phosphorylation of pyroglutamyl peptidase II in Y-79 human retinoblastoma cells after exposure to phorbol ester.

Pyroglutamyl peptidase II (EC 3.4.19.-), a membrane-bound metalloproteinase, is a highly specific TRH-degrading enzyme. Exposure of Y-79 human retinoblastoma cells to 12-0-tetradecanoyl phorbol 13-acetate (TPA) decreased the activity of this enzyme in a time- and concentration-dependent manner (IC50 5 x 10(-9) M). After 15 min of TPA treatment, only 10% of pyroglutamyl peptidase II activity remained. TPA treatment did not affect the activity of the cytosolic enzyme pyroglutamyl peptidase I (EC 3.4.19.3) or the membrane-bound enzyme dipeptidyl peptidase IV (EC 3.4.19.3). Pretreatment of the cells with the protein kinase C inhibitors H-7 or sphingosine prevented the inactivation of pyroglutamyl peptidase II by TPA. The time course of the TPA-mediated effect paralleled the time course of translocation and activation of protein kinase C in this cell line. Immunoblot analysis demonstrated that inactivation of pyroglutamyl peptidase II was not due to dissociation or internalization of this enzyme molecule. Incubation of TPA-activated Y-79 cell membranes with gamma-[32P]-ATP followed by immunoprecipitation revealed a time-dependent phosphorylation of a 48 kilodalton subunit of pyroglutamyl peptidase II. These studies indicate that the phorbol ester effect is mediated by protein kinase C, and reveal a mechanism of potentiation of the action of TRH at its target sites.

Occurrence of pyroglutamyl peptidase II, a specific TRH degrading enzyme in rabbit retinal membranes and in human retinoblastoma cells.

Pyroglutamyl peptidase II, a highly specific thyrotropin releasing hormone (TRH)-degrading enzyme is found in highest concentration in brain where it is localized to synaptic membranes. Retina contains relatively high concentrations of both immunoreactive TRH and TRH receptors. We report that the specific activity of pyroglutamyl peptidase II in rabbit retinal membranes exceeds that of all non-CNS tissues thus far studied. Nine clonal cell lines were screened for this enzymatic activity. The specific activity of pyroglutamyl peptidase II in Y79 retinoblastoma cells was greater than the highest activity found in other cell lines by approximately one order of magnitude. These studies further support a functional relationship between pyroglutamyl peptidase II and TRH and identify a cell line suitable for studies on the regulation of this enzyme.

Metabolism of thyrotropin-releasing hormone in human cerebrospinal fluid. Isolation and characterization of pyroglutamate aminopeptidase activity.

Pyroglutamate aminopeptidase, which catalyzes metabolism of thyrotropin-releasing hormone (TRH) to cyclo(His-Pro), is the major enzyme of TRH metabolism in human CSF. The partially purified CSF pyroglutamate aminopeptidase has a pH optimum between 6.0 and 7.4, and a Km of 15.9 +/- 3.1 microM. A number of potential competitive inhibitors of the enzymatic activity were examined, of which luteinizing hormone-releasing hormone and bombesin were the most effective. An examination of the structure of various peptides that inhibit pyroglutamate aminopeptidase activity indicated that the enzyme generally prefers a substrate having amino-terminal pyroglutamic acid (pGlu) and a COOH-terminal that is either blocked or distant from amino-terminal pGlu. Heavy metals, EDTA and reducing agents inactivated the enyzme, whereas benzamidine, phenylmethylsulfonylfluoride, trypsin inhibitor and alkylating agents had little or no effect on the enzymatic activity. Thiol-oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid), however, considerally inhibited the enzymatic activity. We hypothesize that CSF pyroglutamate aminopeptidase may play a role in the biologic actions of TRH.

Purification of and kinetic studies on a narrow specificity synaptosomal membrane pyroglutamate aminopeptidase from guinea-pig brain.

A pyroglutamate aminopeptidase activity, distinct from that of cytoplasm, was released from a synaptosomal membrane preparation of guinea-pig brain by papain treatment. This activity was further purified 3560-fold relative to the homogenate with a yield of 17% by a procedure involving gel filtration chromatography, calcium phosphate cellulose chromatography and hydrophobic interaction chromatography on phenyl-Sepharose CL-4B. The purified synaptosomal pyroglutamate aminopeptidase hydrolysed only thyroliberin, acid-thyroliberin, the luliberin N-terminal tripeptide (Glp-His-Trp) and, only slightly, Glp-His-Gly. No hydrolysis was observed with dipeptides containing N-terminal pyroglutamic acid (Glp) or with pyroglutamyl peptides containing more than three amino acids. A Km value of 40 microM was recorded when thyroliberin was used as substrate; however, luliberin was found to inhibit the hydrolysis of thyroliberin competitively with a Ki value of 20 microM.

Localization of a narrow-specificity thyroliberin hydrolyzing pyroglutamate aminopeptidase in synaptosomal membranes of guinea-pig brain.

In this paper we report the presence of a particulate pyroglutamate aminopeptidase in guinea-pig brain tissue. This activity appears to reside in the synaptosomal membrane and could be released from the membrane by treatment with papain or Triton X-100. By contrast with a previously described broad-specificity soluble pyroglutamate aminopeptidase from guinea-pig brain tissue, the enzyme released from the synaptosomal membrane preparation removed pyroglutamic acid from thyroliberin, acid thyroliberin and less than Glu-His-Gly alone of the peptides tested. Unlike the soluble tissue enzyme the present enzyme was inhibited by the presence of EDTA and the activity released from synaptosomal membranes by papain was found to have a relative molecular mass of 230 000, almost one order of magnitude greater than that reported for the soluble enzyme.