Purification and characterization of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis

Gonzalès, Th.; Awade, Abalo; Besson, Christine; Robert‐Baudouy, Janine

doi:10.1016/0378-4347(92)80014-h

Cited by 16 publications

(8 citation statements)

References 19 publications

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“…It should be noted angiotensinogen (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), PZ-P-L-G-P-D-R and insulin β-chain eluted at almost the same positions although they eluted separately on an ODS column by RPC (data not shown). It should be noted angiotensinogen (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), PZ-P-L-G-P-D-R and insulin β-chain eluted at almost the same positions although they eluted separately on an ODS column by RPC (data not shown).…”

Section: Resultsmentioning

confidence: 93%

Separation of Amino Acids and Peptides by High Performance Hydrophobic Interaction Chromatography

Yamasaki¹,

Uchida

Tomizawa

et al. 1999

Journal of Liquid Chromatography & Related Technologies

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Chromatographic behaviors of amino acids and peptide derivatives were examined by high performance hydrophobic interaction chromatography(HIC) on two different packings, TSKgels Phenyl-5PW and Ether-5PW. Most amino acids hardly retained and eluted at an elution volume being the same as the column volume even with an initial buffer for hydrophobic interaction chromatography; aromatic amino acids showed weak retention. On the other hand, peptides retained in the column and their elution was roughly in the order of the molecular mass, although some of them showed extraordinary elution behaviors. Kunitz bovine pancreas trypsin inhibitor showed weak retention in spite of having larger molecular mass, which might be due to the steric conformation. Downloaded by [University of Western Ontario] at 10:44 16 November 2014 ORDER REPRINTS 1966 YAMASAKI ET AL.Some peptides showed considerably stronger retention even under ordinary conditions, and the addition of organic modifier (20 % acetonitrile) in eluent was required for their elution. HIC using less hydrophobic packings examined was found to be adequate to separate peptides quantitatively, and this might be useful in removing peptides from contaminants of amino acids and proteins.

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Section: Resultsmentioning

confidence: 93%

Separation of Amino Acids and Peptides by High Performance Hydrophobic Interaction Chromatography

Yamasaki¹,

Uchida

Tomizawa

et al. 1999

Journal of Liquid Chromatography & Related Technologies

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“…The enzyme activity was essentially inhibited by thiol-blocking agents (iodoacetamide, N-ethylmaleimide, and p-chloromercuribenzoate), by diethylpyrocarbonate (a reagent that has been used as a relatively specific probe for histidine residues [28]), and by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide at pH 5.5. For this last one, the trend of decreased modification at increasing pH is typical of carboxyl group modification and is atypical of tyrosine or lysine modification (27) (19). In addition, catalytic activity of the enzyme for pyroglutamyl peptides was confirmed by thin-layer chromatography with Pyr-Ala, PyrAsn-Gly, and Pyr-His-Gly (data not shown).…”

Section: Methodsmentioning

confidence: 85%

“…Phone: (12). Overexpression of these enzymes in Escherichia coli allows simple purification (4,19,45) and makes them suitable as N-terminal pyroglutamyl-unblocking reagents.In order to define more precisely the conserved amino acid domains essential for activity in Pcp enzymes, the present work describes the nucleotide sequence of a pcp gene from a gram-negative bacterial species, Pseudomonas fluorescens. The purification and characterization of the corresponding protein expressed in E. coli are also presented.…”

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confidence: 99%

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Characterization of the pcp gene of Pseudomonas fluorescens and of its product, pyrrolidone carboxyl peptidase (Pcp)

Gonzalès

Robert‐Baudouy

1994

J Bacteriol

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The gene pcp, encoding pyrrolidone carboxyl peptidase (Pcp), from Pseudomonasfluorescens MFO was cloned and its nucleotide sequence was determined. This sequence contains a unique open reading frame (pcp) coding for a polypeptide of 213 amino acids (Mr 22,441) which has significant homology to the Pcps from Streptococcus pyogenes, Bacillus subtilis, and Bacilus amyloliquefaciens. Comparison of the four Pcp sequences revealed two highly conserved motifs which may be involved in the active site of these enzymes. The cloned Pcp from P. fluorescens was purified to homogeneity and appears to exist as a dimer. This enzyme displays a Michaelis constant of 0.21 mM with L-pyroglutamyl-,1-naphthylamide as the substrate and an absolute substrate specificity towards N-terminal pyroglutamyl residues. Studies of inhibition by chemical compounds revealed that the cysteine and histidine residues are essential for enzyme activity. From their conservation in the four enzyme sequences, the Cys-144 and His-166 amino acids are proposed to form a part of the active site of these enzymes.Pyrrolidone carboxyl peptidases (Pcps) (EC 3.4.11.8) are aminopeptidases that selectively remove amino-terminal Lpyroglutamic acid (Pyr) from peptides and proteins (15). These enzymes are present in many bacteria (30) and also in plant, animal, and human tissues (40).Preliminary studies of Pcps have consisted of purification of these enzymes from different bacterial strains (1,24,39,41,43) with a view to using them in protein sequencing to unblock proteins and polypeptides with an N-terminal pyroglutamyl residue prior to sequential Edman degradation (16). These bacterial Pcps, which are cysteine peptidases, showed an absolute specificity for pyroglutamyl residues located at the N terminus of peptides (41, 43) and for chromogenic substrates, such as L-pyroglutamyl-,B-naphthylamide (Pyr-3NA) (41), pyroglutamyl-p-nitroanilide, and pyroglutamyl-4-methylcoumarinylamide (17).Mammalian Pcps were later analyzed and shown to be involved in hormonal and neuronal metabolism (6,8,44). Two different types of Pcps have been detected in mammals. The first, which is a metallopeptidase, is specific for the pyroglutamyl-neuropeptide thyrotropin-releasing hormone (Pyr-HisPro-NH2) and is responsible for regulating the concentration of this neuropeptide (13,31,32). The second is a cysteine peptidase with a general specificity for N-terminal pyroglutamyl residues (7, 10). This Pcp, the function of which has not yet been determined, seems to be more closely related to bacterial Pcps than the first with respect to size, substrate specificity, and sensitivity to inhibitors.Recently, the structural genes encoding Pcps from the gram-positive bacteria Streptococcus pyogenes (12), Bacillus subtilis (3), and Bacillus amyloliquefaciens (45) have been cloned and characterized. The three deduced amino acid sequences are strongly homologous and display long conserved domains. The absence of homology with other peptidases suggests that these Pcps belong to a new class of peptidases * ...

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“…After electrophoretic transfer of the proteins, nitrocellulose membranes were blocked for 2 h at 37ЊC by immersion in 3% gelatin in Tris-buffered saline (TBS) (30 mM Tris-HCl, 150 mM NaCl; pH 7.2). They were incubated for 2 h at room temperature with rabbit anti-S. pyogenes Pcp antibody (13), which had been purified as previously described (28) and diluted in TBS containing 0.05% Tween and 1.0% gelatin. After subsequent washing, the membranes were incubated in the same buffer containing peroxidase-conjugated goat anti-rabbit immunoglobulin G. Serological reactions were detected with a bioluminescent enhanced chemiluminescence (ECL) kit (Amersham).…”

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Mutational analysis of the active site of Pseudomonas fluorescens pyrrolidone carboxyl peptidase

Saux¹,

Gonzales²,

Robert‐Baudouy³

1996

J Bacteriol

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On the basis of chemical inhibition studies and a multiple alignment of four pyrrolidone carboxyl peptidase (Pcp) amino acid sequences, seven conserved residues of the Pseudomonas fluorescens Pcp, which might be important for enzyme activity, have been modified by site-directed mutagenesis experiments. Wild-type and mutant Pcps were expressed in Escherichia coli, purified, and characterized by the ability to cleave the synthetic chromogenic substrate pyroglutamyl-␤-naphthylamide and the dipeptide pyroglutamyl-alanine. Substitution of Glu-10 and Glu-22 by Gln led to enzymes which displayed catalytic properties and sensitivities to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide similar to those of the wild-type Pcp. These residues are not essential for the catalytic activity. Replacement of Asp-89 by Asn and Ala resulted in enzymes which retained nearly 25% of activity and which had no activity, respectively. Substitution of the Cys-144 and His-166 residues by Ala and Ser, respectively, resulted in inactive enzymes. Proteins with changes of Glu-81 to Gln and Asp-94 to Asn were not detectable in crude extract and were probably unstable in bacteria. Our results are consistent with the proposal that Cys-144 and His-166 constitute the nucleophilic and imidazole residues of the Pcp active site, while residue Glu-81, Asp-89, or Asp-94 might constitute the third part of the active site. These results lead us to propose Pcps as a new class of thiol aminopeptidases.

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Purification and characterization of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis

Cited by 16 publications

References 19 publications

Separation of Amino Acids and Peptides by High Performance Hydrophobic Interaction Chromatography

Separation of Amino Acids and Peptides by High Performance Hydrophobic Interaction Chromatography

Characterization of the pcp gene of Pseudomonas fluorescens and of its product, pyrrolidone carboxyl peptidase (Pcp)

Mutational analysis of the active site of Pseudomonas fluorescens pyrrolidone carboxyl peptidase

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