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。我们首先在化学成分确定的培养基中培养金黄色葡萄球菌，并通过使用放射性标记的甲硫氨酸对atl基因产物进行脉冲/脉冲实验。根据使用抗ATL抗体的免疫沉淀的结果，清楚的是ATL作为138 kDa蛋白质分泌，然后加工成115 kDa、85 kDa、62 kDa和51 kDa蛋白质。这些结果表明ATL在跨膜转运后，经历了翻译后加工。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，并将它们与从培养上清液中纯化的那些进行比较。因此，从细胞相关蛋白质提取物中纯化的62 kDa和51 kDa蛋白质显然是62 kDa N-乙酰胞壁酰-L-丙氨酸酰胺酶和51 kDa内切-β-N-乙酰葡糖胺糖苷酶。此外，使用抗ATL抗体的电子显微镜观察显示有两种类型的all基因产物，一种用3 M LiCl可提取，另一种用3 M LiCl不可提取。成熟的ATL如51 kDa氨基葡萄糖苷酶和62 kDa酰胺酶在LiCl可提取组分中占优势，138 kDa蛋白在LiCl不可提取组分中富集。这些结果表明atl基因产物在易位后与某些未知因子结合，经过加工后释放到铜溶液中。我们研究了与ATL相互作用的因子，但只发现它不是蛋白质。该因子的纯化不成功。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.