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Infection and Immunity, September 2003, p. 5238-5244, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.5238-5244.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of an Immunogenic Aminopeptidase of Brucella melitensis

Araceli Contreras-Rodriguez,1 Bernardo Ramirez-Zavala,1 Andrea Contreras,2 Gerhardt G. Schurig,2 Nammalwar Sriranganathan,2 and Ahide Lopez-Merino1*

Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, México,1 Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia 24061-03422

Received 9 April 2003/ Returned for modification 8 May 2003/ Accepted 18 June 2003

An immunogenic aminopeptidase was purified from Brucella melitensis strain VTRM1. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps. This procedure resulted in a yield of 29% and a 144-fold increase in specific activity. The aminopeptidase appeared to be a monomeric enzyme with a molecular mass of 96 kDa and an isoelectric point of 4.8. Its activity was optimal at pH 7.0 at 40°C. The enzyme was strongly inhibited by EDTA, 1,10-phenathroline, and divalent cations (Zn2+ and Hg2+), suggesting that this protein was a metalloaminopeptidase. The enzyme showed preference for alanine at the N termini of aminoacyl derivatives. The Km values for L-alanine-p-nitroanilide (Ala-pNA) and Lys-pNA were 0.35 and 0.18 mM, respectively. The N-terminal sequence of aminopeptidase was used for a homologous search in the genomes of B. melitensis 16M and Brucella suis 1330. The analysis revealed an exact match of the probe sequence (36 bp) with an open reading frame of 2,652 bp encoding a protein predicted to be alanyl aminopeptidase (aminopeptidase N). Collectively, these data suggest designation of the B. melitensis enzyme as an aminopeptidase N. The aminopeptidase was recognized by sera from patients with acute and chronic brucellosis, suggesting that the enzyme may have important diagnostic implications.

* Corresponding author. Mailing address: Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Apartado Postal CON 174, México D.F., C.P. 06400, México. Phone: 5255 5729-6209. Fax: 5255 5729-6207. E-mail: ahide99{at}hotmail.com.

Editor: D. L. Burns

Infection and Immunity, September 2003, p. 5238-5244, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.5238-5244.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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