Waurzyniak B J, Clinkenbeard K D, Confer A W, Srikumaran S
Department of Veterinary Pathology, Oklahoma State University, Stillwater 70478.
Am J Vet Res. 1994 Sep;55(9):1267-74.
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.
溶血巴斯德菌A1在含有0.5%牛血清白蛋白（BSA）的RPMI 1640培养基中培养2.5小时后，培养上清液的白细胞毒性活性增强（30,700±12,900毒性单位/毫升，相比之下，仅在RPMI 1640培养基中产生的培养上清液的白细胞毒性活性为120±40毒性单位/毫升）。在含有50 mM氯化钠的缓冲液中，对RPMI 1640培养基上清液的白细胞毒性活性进行凝胶过滤色谱分析，结果显示一个单一的白细胞毒性活性峰（峰I）在接近凝胶树脂分子量排阻极限（估计分子量约为8,000 kd）处洗脱。相比之下，在RPMI 1640加牛血清白蛋白培养基（RPMI + BSA）中产生的培养上清液有峰I以及另外两个白细胞毒性活性峰（峰II和峰III），估计分子量分别约为80 kd和<30 kd。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以及用抗白细胞毒素的单克隆抗体进行免疫印迹分析确定，所有白细胞毒性活性峰均由分子量约为100 kd的白细胞毒素原体组成。将RPMI + BSA中产生的培养上清液白细胞毒性活性在含有500 mM氯化钠或6M尿素的缓冲液中进行凝胶过滤色谱分析，结果分别检测到仅一个估计分子量约为250 kd和800 kd的单一白细胞毒性活性峰。这些发现表明，溶血巴斯德菌以高分子量聚集体形式存在，白细胞毒性活性较低，在BSA存在的情况下，部分解聚为多种具有增强白细胞毒性活性的毒素形式。其中一些白细胞毒素形式在低离子强度下与基于葡聚糖的凝胶树脂相互作用。
