| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 97 === Abstract
Xanthomonas campestris pv. campestris utilizes the type II secretion system for secreting extracellular hydrolytic enzymes to degrade the surface structure of plant cells. The secretion apparatus is composed of 12 gene products that are thought to form a multiprotein complex, which spans the periplasmic compartment. It is specifically involved in translocation of the secreted proteins across outer membrane. Of all protein components, XpsE is the only cytoplasmic protein that lacks any membrane spanning region. There are four essential nucleotide binding motifs located within the C domain of XpsE, which is made capable of ATP binding and hydrolyzing activities. As suggested from previous studies, XpsE oligomerizes into mutilmer before it interacts with the N domain of XpsL, an integral membrane protein. It has been proposed that XpsE may play an important role as energy supplier and/or signal transmitter. In order to gain better understanding about the molecular mechanism of how XpsE hydrolyzes ATP, I made use of HPLC for identifying protein-bound nucleotide. Following purification through affinity column, XpsE or XpsE/MBP-XpsLN complex was boiled to release the protein-bound nucleotide and separated through HPLC. As suggested from the HPLC profile, no significant signal that is above the machine detection limit was detected at the ADP/ATP retention time. To examine if bound nucleotide could be detected by incubating the XpsE/MBP-XpsLN with exogenously added ATP, I incubated the protein complex with ATP and Mg2+ on ice for 15 min followed by passing through the G25 Sepharose for the removal of free nucleotides and boiling for the release of protein-bound nucleotide before HPLC analysis. There was still no detectable protein-bound ATP/ADP. Unexpectedly, I found that the added ATP was hydrolyzed by the protein complex at 4 ºC. Within 15 minutes, about 40 % of the added ATP was converted into ADP. Moreover, such ATP hydrolysis activity was inhibited by vanadate. Further studies indicated that at the molecular ratio of XpsE to ATP of 1:13, ATP hydrolysis seems to reach its maximal level within a very short period of time. In summary, XpsE or XpsE/MBP-XpsLN complex, when purified from affinity column, may stay in a state with no bound or only trace amount of ATP/ADP. Although the added ATP could be hydrolyzed at 4 ºC, there was still no detectable protein-bound ATP/ADP implying the ADP generated from ATP hydrolysis by the XpsE/MBP-XpsLN was released from XpsE soon after the ATP was hydrolyzed.
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