REGULATION MECHANISM OF STAPHYLOCOCCUS AUREUS LYTIC ENZYME

本课题旨在了解金黄色葡萄球菌溶菌酶的调控机制。我们首先在化学确定的培养基中培养金黄色葡萄球菌，并利用放射性标记的蛋氨酸进行atl基因产物的脉冲/追赶实验。根据抗ATL抗体免疫沉淀的结果，ATL以138 kDa蛋白的形式分泌，然后加工成115 kDa、85 kDa、62 kDa和51 kDa蛋白。这些结果表明，ATL在细胞膜上易位后发生翻译后加工。我们还纯化了与金黄色葡萄球菌细胞相关的所有基因产物，并将其与培养上清纯化的基因产物进行了比较。因此，从细胞相关蛋白提取物中纯化的62 kDa和51 kDa蛋白显然是62 kDa n -乙酰muramyl- l-丙氨酸氨基酶和51 kDa内切- β - n -乙酰氨基葡萄糖苷酶。此外，利用抗atl抗体电镜观察发现，ail基因产物有两种类型，一种是用3M LiCl可提取的，另一种是用3M LiCl不可提取的。成熟ATL如51 kDa氨基葡萄糖酶和62 kDa氨基酶在licl可提取部分中占主导地位，138 kDa蛋白在licl不可提取部分中丰富。这些结果表明，atl基因产物在易位后与某种未知因子结合，经过加工并释放到铜离子中。我们研究了与ATL相互作用的因素。但才发现它不是蛋白质。该因子的纯化未成功。它的身份仍然未知。

The aim of this project was to understand regulation mechanism of Staphylococcus aureus bacteriolytic enzyme. We first grew S.aureus in chemically defined medium and performed pulsc/chasc experiment of atl gene product by using radio-labeled methionine. According to the results of immunprecipitation using anti-ATL antibody, it was clear that ATL was secreted as 138 kDa protein, and thereafter processed to 115 kDa, 85 kDa, 62 kDa and 51 kDa proteins. These results showed that ATL undergo posttranslational processing after it is translocated the cell membrane. We also purified ail gene products associated with S.aureus cells, and compared them with those purifed from culture supernatant. Accordingly, it became apparent that 62 kDa and 51 kDa proteins purified from extract of cell-associated protein were 62 kDa N-acetylmuramyl-L-alanine amidase and 51 kDa endo-beta-N-acetylglucosaminidase. Furthermore, electron microscopical observation using anti-ATL antibody revealed that there are two types of ail gene products, the one extractable with 3M LiCl and the other nonextractable with 3M LiCl. Matured ATL such as 51 kDa glucosaminidase and 62 kDa amidase are predominat in LiCl-extractable fraction and 138 kDa protein was rich in LiCl-nonextractable fraction. These results sugest that atl gene products associate with some unknown factor after it is translocated, and undergo processing and are released into cupernatant. We investigated the factor which interact with ATL.but only found that it is not protein. Purification of the factor was unsuccessful. and its identity remains unknown.