Clinkenbeard K D, Clinkenbeard P A, Waurzyniak B J
Department of Veterinary Pathology, College of Veterinary Medicine, Oklahoma State University, Stillwater 74078, USA.
Vet Microbiol. 1995 Jul;45(2-3):201-9. doi: 10.1016/0378-1135(94)00131-f.
Pasteurella haemolytica biotype A, serotype 1 grown to late logarithmic growth phase in cell culture medium (RPMI 1640) produced highly aggregated leukotoxin. The multimer mass of the highly aggregated leukotoxin in 0.1 M sodium phosphate buffer pH 7.0 as determined by gel filtration chromatography on Sephacryl S400HR was approximately 8000 kDa. Resuspension of leukotoxin in phosphate buffer containing various chaotropic agents resulted in partial disaggregation of leukotoxin and enhanced leukotoxic activity. 3M guanidine disaggregated leukotoxin to a multimer mass of approximately 800 kDa and enhanced leukotoxic activity 3 to 20-fold. In 6 M urea or 1 M sodium thiocyanate, leukotoxin multimers were observed ranging in mass from 8000 kDa to 400 kDa, and activity enhancement was less than that for leukotoxin in 3 M guanidine. Several detergents were tested for enhancement of leukotoxic activity, but only 1% Tween 20 enhanced leukotoxic activity (4-fold), whereas 1.25% octylglucoside, 10 mM CHAPS, and 5 mM deoxycholate diminished and 1% Triton X-100 abolished leukotoxic activity.
溶血巴斯德氏菌生物型A，血清型1在细胞培养基（RPMI 1640）中生长至对数生长后期，产生高度聚集的白细胞毒素。通过在Sephacryl S400HR上进行凝胶过滤色谱法测定，在pH 7.0的0.1 M磷酸钠缓冲液中高度聚集的白细胞毒素的多聚体质量约为8000 kDa。将白细胞毒素重悬于含有各种离液剂的磷酸盐缓冲液中，导致白细胞毒素部分解聚并增强了白细胞毒性活性。3 M胍将白细胞毒素解聚为多聚体质量约为800 kDa，并使白细胞毒性活性增强3至20倍。在6 M尿素或1 M硫氰酸钠中，观察到白细胞毒素多聚体的质量范围为8000 kDa至400 kDa，活性增强程度低于3 M胍中的白细胞毒素。测试了几种去污剂对白细胞毒性活性的增强作用，但只有1%吐温20增强了白细胞毒性活性（4倍），而1.25%辛基葡糖苷、10 mM CHAPS和5 mM脱氧胆酸盐降低了白细胞毒性活性，1% Triton X-100则消除了白细胞毒性活性。
