Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae.
Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae.
复制标题
酿酒酵母野生型和糖酵解途径突变体的 Phosphorus-31 核磁共振研究。
作者：
G. Navon;R. Shulman;T. Yamane;T. Eccleshall;K. Lam;J. J. Baronofsky;J. Marmur
在145.7 MHz频率下，对酿酒酵母野生型和突变型菌株的高分辨~(31)P核磁共振谱进行了观察。研究了在好氧和厌氧条件下添加葡萄糖时不同磷代谢产物的水平。分离到3株突变菌株，其生化缺陷表现为：PFK缺乏磷酸果糖激酶活性，PGI缺乏磷酸葡萄糖异构酶活性，CIF没有葡萄糖分解代谢抑制果糖二磷酸酶活性。当向细胞悬浮液中加入葡萄糖时，发现每个突变菌株都积累了特有的糖磷酸盐。在磷酸果糖激酶缺失突变体的情况下，观察到了戊糖分流代谢物的出现。用pH滴定的方法对细胞的酸抽提物进行了31P核磁共振峰的指认。细胞内多聚磷酸盐中末端、倒数第二个和中心磷原子的独立信号允许估计它们的平均分子量。可以观察到甘油(3)磷酸、甘油(3)磷酸丝氨酸、甘油(3)磷乙醇胺以及三种核苷酸二磷酸糖的信号。静息和厌氧细胞内pH在6.5~6.8之间，腺苷3‘-磷酸(ATP)水平较低。通入氧气后，细胞内的三磷酸腺苷水平显著增加，细胞内的pH值达到pH 7.2--7.3，表明这些细胞内的质子转运活跃。在适当的条件下，可以检测到一个代表细胞器之一的无机磷的新的峰，该细胞器的pH与细胞质的pH不同。
High-resolution phosphorus-31 nuclear magnetic resonance (31P NMR) spectra of wild-type and mutant strains of Saccharomyces cerevisiae were observed at a frequency of 145.7 MHz. Levels of various phosphorus metabolites were investigated upon addition of glucose under both aerobic and anaerobic conditions. Three mutant strains were isolated and their biochemical defects characterized: pfk lacked phosphofructokinase activity; pgi lacked phosphoglucose isomerase activity; and cif had no glucose catabolite repression of the fructose bisphosphatase activity. Each mutant strain was found to accumulate characteristic sugar phosphates when glucose was added to the cell suspension. In the case of the phosphofructokinase deficient mutant, the appearance of a pentose shunt metabolite was observed. 31P NMR peak assignments were made by a pH titration of the acid extract of the cells. Separate signals for terminal, penultimate, and central phosphorus atoms in intracellular polyphosphates allowed the estimation of their average molecular weight. Signals for glycero(3)phosphochline, glycero(3)phosphoserine, and glycero(3) phosphoethanolamine as well as three types of nucleotide diphosphate sugars could be observed. The intracellular pH in resting and anaerobic cells was in the range 6.5--6.8 and the level of adenosine 5'-triphosphate (ATP) low. Upon introduction of oxygen, the ATP level increased considerably and the intracellular pH reached a value of pH 7.2--7.3, irrespective of the external medium pH, indicating active proton transport in these cells. A new peak representing the inorganic phosphate of one of the cellular organelles, whose pH differed from the cytoplasmic pH, could be detected under appropriate conditions.