Search For The Target Of Ethambutol By Proteomics Method

The tuberculosis is one of the main threats of the human health。Atpresent, the tubercle bacillus' drug-resistance phenomenon is getting moreand more serious and has had weakened the curative effect of the presentantituberculotic greatly. The severe situation demands to accelerate theresearch and development of new types of tuberculosis medicine. Ethambutol is a bacteriostat, and is effective only to extracellularreproductive period bacterium. For a long time, the effect mechanism ofEthambutol has been considered being able to cast effect onarabinoglycosyltransferase. arabinoglycosyltransferase participates thebiosynthesis of arabinogalactan in the cell-wall, and by linking araban inthe Decaprenyl phosphate-Ara(DPA)to a specified position of galactan link,it produces branched arabinogalactan structure. Ethylaminobutanol maycombine with arabinoglycosyltransferase competitively to refrain thetransfer of araban, and thus influence the formationof mycolic acid-arabinogalactan –peptidoglycan compound in the cell wall. This has madeit easier for other medicine such as rifampicin to enter cells so as toproduce the effect of anti-tuberculosis together. Prof. Mike McNeil, who is from Microbiology Department ofColorado State University, US, conducted an experiment which showedthat if add Ethambutol into culture medium, a great amount ofarabinogalactan in cell-wall lose in a short a period of time. This seemstohave certain correlation with the increase of hydrolase. 3 The target of new medicine can be found by the proteomics method.The major proteomics research method is 2-D electrophoresis, which is ofhigh degree of sensitivity and low cost, and able to detect subtle changes ofboth known and unknown protein. Based on differentisoelectric focusingways, 2-D electrophoresis can be divided into IPG-DALT 和 ISO-DALT.The advantage of IPG-DALT lies in high stability of pH gradient, highresolution, high reproductivity,and high loading volume. Whereas itsdisadvantage is that IPG strip is expensive and requires high qualityinstruments and equipments.Though ISO-DALT suffers from lowerresolution, reproductivity and stability compared with IPG-DALT, it is easyto operate, less expensive and therefore easy to be popularized.Furthermore, ISO-DALT has achieved satisfactory resolution, stability andreproductivity after the experiment conditions are optimized. We found 5and 11 protein spot whose expression changed, respectively in the twoexperiments where ISO-DALT is used. And these protein spot can behydrolyze into peptide with trypsin; then analyze the result with massspectrum. After data base retrieve and analysis, we considered that one ofthe protein might be athiol peroxidase (TPX). In order to discover the target of Ethambutol on AG, we adoptedHPLC method to analyze the changes of arabinan structure. And we foundthat after treated by Ethambutol, non-reducing end of hexasaccharide fromarabinan decreases, suggesting that the target of Ethambutol might bebranched non-reducing end of arabinan, but not the linear part of thearabinan molecules.