Purification and characterization of an immunogenic aminopeptidase of Brucella melitensis.
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 degrees C. The enzyme was strongly inhibited by EDTA, 1,10-phenathroline, and divalent cations (Zn(2+) and Hg(2+)), suggesting that this protein was a metalloaminopeptidase. The enzyme showed preference for alanine at the N termini of aminoacyl derivatives. The K(m) 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.
Contreras-Rodriguez
Araceli
A
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, México.
Ramirez-Zavala
Bernardo
B
Contreras
Andrea
A
Schurig
Gerhardt G
GG
Sriranganathan
Nammalwar
N
Lopez-Merino
Ahide
A
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D000907:Antibodies, Bacterial / Q000097:blood; D017347:Brucella melitensis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D002006:Brucellosis / Q000175:diagnosis / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D000907:Antibodies, Bacterial; D011480:Protease Inhibitors; D000626:Aminopeptidases