RESUMO
A hybridoma cell line producing a rat monoclonal antibody (MAb Bo-33) directed against lipopolysaccharide of Azospirillum brasilense Wa5 has been established and characterized. Whole bacteria were used as immunogens. The number of antigens per cell was about 1500. The number of antigens per cell of reisolates from the rhizosphere of what was similar to the number of antigens of bacteria cultivated in rich medium. The sensitivity of detection using MAb Bo-33 was about 100 bacteria/ml. Therefore, the MAb was suitable for in situ immunofluorescence detection and a sensitive direct quantification of Azospirillum brasilense Wa5 in rhizosphere extracts.
Assuntos
Anticorpos Antibacterianos/imunologia , Azospirillum brasilense/imunologia , Lipopolissacarídeos/imunologia , Raízes de Plantas/microbiologia , Microbiologia do Solo , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Azospirillum brasilense/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Epitopos , Imunofluorescência , Hibridomas , Lipopolissacarídeos/isolamento & purificação , Ratos , Triticum/microbiologiaRESUMO
1. Adamalysin II, alias proteinase II, a 24-kDa zinc-endopeptidase from the snake venom of Crotalus adamanteus, is a member of a large family of metalloproteinases isolated as small proteinases or proteolytic domains of mosaic hemorrhagic proteins from various snake venoms. Homologous domains have been recently detected in multimodular mammalian reproductive tract proteins and in mammalian gene products, somatic rearrangements of which seem to be linked to primary breast cancers. 2. The 2.0 A X-ray crystal structure of adamalysin II reveals an ellipsoidal molecule with a shallow active-site cleft separating a relatively irregularly folded sub-domain from the main molecular body composed of a 5-stranded beta-sheet and four alpha-helices. Opposite to this active-site cleft is an integrated calcium ion liganded by carbonyl and strongly conserved carboxylate/carboxamide residues. The folding of the peptide fragment containing the zinc-binding motif HExxHxxGxxH bears only a distant resemblance to thermolysin; it is identical to that found in astacin, in collagenases, and in serralysins, with the three histidines (His142, His146, His152) and a water molecule (linked to the glutamic acid Glu143) likewise constituting the zinc ligand; similar to collagenases, but in contrast to astacin, adamalysin II lacks a fifth (tyrosine) zinc ligand, leaving its zinc-ion tetrahedrally coordinated. Furthermore, adamalysin II shares an identical active-site basement formed by a common Met-turn. 3. Due to their virtually identical active-site environment and similar folding topology, the snake venom metalloproteinases (hitherto called adamalysins) and the three other proteinases might be grouped into a common superfamily called metzincins with distinct differences from the thermolysin family.