| Summary: | Each of two affinity isolation methods, the first based on biotinylated porcine transferrin plus streptavidin-agarose, and the second on Sepharose-coupled porcine transferrin, followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), allowed the isolation and identification of two potential porcine-transferrin-binding polypeptides (∼64 kDa and 99 kDa) from total membranes of Actinobacillus pleuropneumoniae grown under iron-restricted conditions. Both polypeptides were iron-repressible and were identified as potential receptor candidates as they were not isolated when biotinylated human transferrin was used instead of biotinylated porcine transferrin. The 64 kDa polypeptide was the more easily removed from Sepharose-coupled porcine transferrin and only the 99 kDa polypeptide appeared to be an outer membrane protein. These results suggest that the 99 kDa polypeptide represents the porcine transferrin receptor of A. pleuropneumoniae, and that the 64 kDa polypeptide represents an associated protein serving an accessory role. Fe+3 uptake studies using plate assays and attempted isolation of a putative lactoferrin receptor using biotinylated porcine lactoferrin plus streptavidin-agarose, followed by SDS-PAGE, showed that A. pleuropneumoniae lacks a mechanism for the use of porcine lactoferrin as an iron source.
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