Growth of Pasteurella haemolytica A 1 in media containing fetal bovine serum has been observed to enhance leukotoxin (LKT) activity, but the mechanism of this increase is not understood. We found that bovine serum albumin in the absence of other serum components also enhances LKT activity. During the logarithmic growth phase, LKT activity was 30,700 � 12,900 Toxic Units (TU)/ml for P. haemolytica grown in RPM I medium containing 0. 5% bovine serum albumin (BSA) (BSA-LKT), compared to 120 � 40 TU/ml in medium containing RPMI alone (RPMILKT). In other experiments, addition of 0.5% BSA to RPMI-LKT culture filtrate(RPMI-LKT/BSA) resulted in LKT activity intermediate ( 13,000 � 1,600 TU/ml) between that of BSA-LKT (55,600 � 11,500 TU/ml) and RPMI-LKT (2,200 � 900 TU/ml). The activity of RPMI-LKT, BSA-LKT and RPMI-LKT /BSA decreased when the toxin preparations were incubated at room temperature (25�C) for 2 hours; however, the decrease in RPMI-LKT activity was more pronounced. Concentrated toxin from logarithmic growth phase RPMI-LKT culture supernatants contained a single LKT activity peak (Peak I) on Sephacryl HR-400 which had a K of 0.01 and estimated av molecular weight > > 669,000. In contrast, gel filtration of concentrated toxin from logarithmic growth phase BSA-LKT culture supernatants had three activity peaks with K values of 0.04 (Peak 1), 0.66 (Peak II), and av 0.87 (Peak Ill). Gel filtration of RPMI-LKT/BSA also consisted of Peaks I, II, and Ill. Peaks I, II, and Ill contain a prominent 97000 band on SDS-PAGE which was identified as LKT by Western blot analysis with an monoclonal antibody (MAB) to LKT. The identity of Peaks I, II, and Ill were further confirmed as LKT by target cell specificity (lysed bovine lymphoma cells and not equine leukocytes) and neutralization with a MAB toP. haemolytica LKT. It is concluded that LKT produced in RPMI medium alone is a large aggregate which undergoes conformational changes in the presence of BSA resulting in at least two additional LKT forms which may be partially disaggregated forms of the large aggregate LKT produced in RPMI.Veterinary Patholog
