INTRACELLULAR-LOCALIZATION OF THE DERMONECROTIC TOXIN OF BORDETELLA-PERTUSSIS
INTRACELLULAR-LOCALIZATION OF THE DERMONECROTIC TOXIN OF BORDETELLA-PERTUSSIS
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DOI：
10.1128/iai.25.3.896-901.1979
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发表时间：
1979-01-01
影响因子：
3.1
通讯作者：
MANCLARK, CR
中科院分区：
文献类型：
--
作者：
COWELL, JL;HEWLETT, EL;MANCLARK, CR
B热不稳定性皮肤坏死毒素的定位。通过使用4日龄乳鼠的皮肤反应作为毒素测定法，研究了在化学成分确定的Scholer-Scholte培养基中生长的百日咳菌株114。通过对数生长期并进入生长稳定期，毒素与细胞结合，在培养上清液中未检测到。在全细胞悬浮液中仅检测到约4%的裂解细胞悬浮液中存在的活性，并且通过使全细胞经受渗透压休克（一种从许多革兰氏阴性细菌的周质空间释放蛋白质的程序）不会释放皮肤坏死活性。在细胞裂解和制备可溶性和膜级分之后，细胞裂解物中73-80%的活性在可溶性级分中回收，仅3-6%存在于膜级分中。皮肤坏死毒素的细胞内细胞质定位的进一步证据是毒素对胰蛋白酶处理全细胞的不敏感性。用胰蛋白酶（80 μ g/ml）在37 ℃处理全细胞20分钟。C没有降低皮肤坏死或苹果酸脱氢酶活性，但抑制了95%以上的细胞质外腺苷酸环化酶活性。相同的胰蛋白酶处理的细胞裂解物降低了所有上述活动的90%以上。
Localization of the heat-labile dermonecrotic toxin of B. pertussis strain 114 grown in chemically defined Stainer-Scholte medium was studied by using skin reactions in 4 day old suckling mice as the assay for toxin. Through log phase and into stationary phase of growth the toxin was cell-associated and not detected in the culture supernatant. Only about 4% of the activity present in a suspension of lysed cells was detected in a suspension of whole cells, and the dermonecrotic activity was not released by subjecting whole cells to osmotic shock, a procedure that releases proteins from the periplasmic space of many gram-negative bacteria. After cell lysis and preparation of soluble and membrane fractions, 73-80% of the activity in the cell lysate was recovered in the soluble fraction, with only 3-6% present in a membrane fraction. Further evidence for the intracellular cytoplasmic localization of the dermonecrotic toxin was the insensitivity of the toxin to trypsin treatment of whole cells. Treatment of whole cells with trypsin (80 .mu.g/ml) for 20 min at 37.degree. C did not decrease dermonecrotic or malate dehydrogenase activities, but did inhibit more than 95% of the extracytoplasmic adenylate cyclase activity. Identical trypsin treatment of a cell lysate decreased all the above activities by more than 90%.