Enhancement of Pasteurella haemolytica leukotoxic activity by bovine serum albumin.
Growth of Pasteurella haemolytica A1 in RPMI 1640 medium containing 0.5% bovine serum albumin (BSA) for 2.5 hours enhanced culture supernatant leukotoxic activity (30,700 +/- 12,900 toxic units/ml, compared with leukotoxic activity of culture supernatants produced in RPMI 1640 medium alone (120 +/- 40 toxic units/ml). Gel filtration chromatography of the leukotoxic activity from RPMI 1640 medium supernatants in buffer containing 50 mM NaCl indicated a single leukotoxic activity peak (peak I) eluting near the gel resin molecular mass exclusion limit (estimated molecular mass of approx 8,000 kd). In contrast, culture supernatants produced in RPMI 1640 plus bovine serum albumin medium (RPMI + BSA) had peak I and 2 additional leukotoxic activity peaks (peaks II and III) with estimated molecular mass of approximately 80 and < 30 kd, respectively. All leukotoxic activity peaks were composed of approximately 100-kd molecular mass leukotoxin protomer, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against leukotoxin. Subjecting culture supernatant leukotoxic activity produced in RPMI + BSA to gel filtration chromatography in buffer containing 500 mM NaCl or 6M urea resulted in detection of only a single leukotoxic activity peak with estimated approximate molecular mass of 250 and 800 kd, respectively. These findings suggest that P haemolytica exists as a high molecular mass aggregate with low leukotoxic activity which, in the presence of BSA, partially disaggregates to multiple toxin forms with enhanced leukotoxic activity. Some of these leukotoxin forms interact with dextran-based gel resins at low ionic strength.
Waurzyniak
B J
BJ
Department of Veterinary Pathology, Oklahoma State University, Stillwater 70478.
Clinkenbeard
K D
KD
Confer
A W
AW
Srikumaran
S
S
D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000633:toxicity; D002417:Cattle; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002850:Chromatography, Gel; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005098:Exotoxins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000633:toxicity; D007770:L-Lactate Dehydrogenase; D016978:Mannheimia haemolytica / Q000254:growth & development* / Q000378:metabolism; D008970:Molecular Weight; D012710:Serum Albumin, Bovine; D014407:Tumor Cells, Cultured
D001427:Bacterial Toxins; D003470:Culture Media; D005098:Exotoxins; C018579:leukotoxin; D012710:Serum Albumin, Bovine; D007770:L-Lactate Dehydrogenase