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Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.
Can J Vet Res. 1998 Jul; 62(3):183-90.CJ

Abstract

A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.

Links

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Authors+Show Affiliations

Negrete-Abascal E
Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, México D.F., Mexico.
Tenorio VR
No affiliation info available
Guerrero AL
No affiliation info available
García RM
No affiliation info available
Reyes ME
No affiliation info available
de la Garza M
No affiliation info available

MeSH

Actinobacillus pleuropneumoniaeAnimalsChromatography, AffinityChromatography, DEAE-CelluloseElectrophoresis, Polyacrylamide GelEndopeptidasesFreeze DryingImmunoglobulin AImmunoglobulin GKineticsMolecular WeightSerotypingSubstrate SpecificitySwineUltrafiltration

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9684047

Citation

Negrete-Abascal, E, et al. "Purification and Characterization of a Protease From Actinobacillus Pleuropneumoniae Serotype 1, an Antigen Common to All the Serotypes." Canadian Journal of Veterinary Research = Revue Canadienne De Recherche Veterinaire, vol. 62, no. 3, 1998, pp. 183-90.
Negrete-Abascal E, Tenorio VR, Guerrero AL, et al. Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes. Can J Vet Res. 1998;62(3):183-90.
Negrete-Abascal, E., Tenorio, V. R., Guerrero, A. L., García, R. M., Reyes, M. E., & de la Garza, M. (1998). Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes. Canadian Journal of Veterinary Research = Revue Canadienne De Recherche Veterinaire, 62(3), 183-90.
Negrete-Abascal E, et al. Purification and Characterization of a Protease From Actinobacillus Pleuropneumoniae Serotype 1, an Antigen Common to All the Serotypes. Can J Vet Res. 1998;62(3):183-90. PubMed PMID: 9684047.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes. AU - Negrete-Abascal,E, AU - Tenorio,V R, AU - Guerrero,A L, AU - García,R M, AU - Reyes,M E, AU - de la Garza,M, PY - 1998/7/31/pubmed PY - 1998/7/31/medline PY - 1998/7/31/entrez SP - 183 EP - 90 JF - Canadian journal of veterinary research = Revue canadienne de recherche veterinaire JO - Can J Vet Res VL - 62 IS - 3 N2 - A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae. SN - 0830-9000 UR - https://www.unboundmedicine.com/medline/citation/9684047/Purification_and_characterization_of_a_protease_from_Actinobacillus_pleuropneumoniae_serotype_1_an_antigen_common_to_all_the_serotypes_ DB - PRIME DP - Unbound Medicine ER -