Calcium binds to and is required for biological activity of the 104-kilodalton hemolysin produced by Actinobacillus pleuropneumoniae serotype 1.
Actinobacillus pleuropneumoniae serotype 1, strain Shope 4074, was grown on agar medium containing 10 mM Ca2+ and under Ca2+ limiting conditions by addition of 2 and 5 mM EGTA to the growth medium. Hemolysis of washed bovine erythrocytes was observed from the culture grown in Ca2+ excess but not from the two cultures where Ca2+ was chelated from the growth medium by using EGTA. However, the hemolytic activity of these latter two cultures could be restored if 10 mM Ca2+ was added to the dilution buffer. This restored hemolytic activity could be neutralized with a rabbit homologous polyclonal antiserum specific for the 104-kDa hemolysin of serotype 1 but not with preimmune serum from the same rabbit. Stained SDS-PAGE gels and immunoblots showed the 104-kDa protein hemolysin in fractions from each of the three growth conditions. Thus, the restored hemolytic activity was associated with the 104-kDa protein in the three cultures. In addition, the 104-kDa protein, when electroblotted onto nylon membranes, bound 45Ca, indicating that the molecule has binding sites for Ca2+. The results indicate that Ca2+ is required for the biological activity of the 104-kDa hemolysin of A. pleuropneumoniae serotype 1.
Devenish
J
J
Department of Veterinary Microbiology and Immunology, University of Guelph, Ont., Canada.
Rosendal
S
S
D000188:Actinobacillus / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006460:Hemolysin Proteins / Q000378:metabolism*; D006461:Hemolysis / Q000187:drug effects*; D015151:Immunoblotting
D003470:Culture Media; D006460:Hemolysin Proteins; D002118:Calcium