摘要
为了分析酿酒酵母在不同培养条件下的代谢调控过程的差异 ,采用固相pH梯度 SDS聚丙烯酰胺双向凝胶电泳对其利用不同碳源时细胞的总蛋白进行了分离 ,银染显色 ,使用 2D蛋白质图像分析系统ImageMaster 2DElite对双向电泳图谱进行分析 ,查询SWISS 2DPAGE蛋白质组数据库 ,识别了约 5 0 0个蛋白质点。对与糖酵解途径、磷酸戊糖途径、三羧酸循环和几种回补反应相关的大部分关键的蛋白质进行了差异分析。探讨了酿酒酵母利用不同碳源时及生长的不同阶段代谢机理的变化和在蛋白质水平的调控。
For the investigation of the metabolic regulation of Saccharomyces cerevisiae under different culture conditions, the proteins of cell utilizing various carbon sources were separated by two-dimensional electrophoresis with immobilized pH gradients as the first dimension and SDS-PAGE as the second. Samples were taken in the log phase of batch culture using glucose or lactic acid as carbon source, while another sample was taken from the broth when glucose was consumed up and ethanol accumulated in the previous phase was further metabolized. After electrophoresis, the protein spots were detected by silver-stain in a Hoefer Automated Gel Stainer with a protein silver staining kit. Silver-stained gels were scanned and digitized to create computer images. About 500 protein spots were detected by employing the 2D proteome image analysis system Image Master 2D Elite and SWISS-2DPAGE proteome database. Most of the protein expressed and involved in the glycolysis, pentose phosphate (PP) pathway, anaplerotic pathway, as well as TCA cycle were analyzed. The metabolism regulation of protein level for Saccharomyces cerevisiae under various carbon sources, as well as during different phase of growth, was studied. The expression of several glycolytic en^zymes(glk, pgi, pgk, eno, pyk) was up-regulated while the expression of enzymes in oxidative pentose phosphate pathway(zwf, gnd) was down-regulated when ethanol and lactic acid were taken as carbon source. Simultaneously, frucotose1,6-biphosphatase was found to be up-regulated due to the gluconeogenic requirement. Citrate synthase and Malate dehydrogenase do not exhibit significant difference, indicating TCA cycle is necessary when utilizing glucose, ethanol or lactic acid as carbon source. Thus, the NADPH loss due to the repressed pentose phosphate pathway could be compensated by TCA cycle in cases of ethanol and lactic acid. The expression of malic enzyme and isocitrate lyase are activated to a large extent when metabolizing ethanol, indicating glyoxylate shunt is essential in transferring ethanol to generate four carbon precursors for the biosynthesis and the NADP-dependent malic enzyme could also serve as compensation mechanism for NADPH loss in this case.
出处
《生物工程学报》
CAS
CSCD
北大核心
2004年第3期398-402,共5页
Chinese Journal of Biotechnology
基金
国家自然科学基金资助 (No .2 0 2 760 65 )~~
关键词
双向凝胶电泳
蛋白质组
代谢调控
酵母
two-dimensional electrophoresis, proteome,metabolic regulation, Saccharomyces cerevisiae
