THIN-LAYER CHROMATOGRAPHIC METHODS TO ISOLATE P-32-LABELED 5-PHOSPHORIBOSYL-ALPHA-1-PYROPHOSPHATE (PRPP) - DETERMINATION OF CELLULAR PRPP POOLS AND ASSAY OF PRPP SYNTHETASE-ACTIVITY
THIN-LAYER CHROMATOGRAPHIC METHODS TO ISOLATE P-32-LABELED 5-PHOSPHORIBOSYL-ALPHA-1-PYROPHOSPHATE (PRPP) - DETERMINATION OF CELLULAR PRPP POOLS AND ASSAY OF PRPP SYNTHETASE-ACTIVITY
复制标题
DOI：
10.1016/0003-2697(79)90138-6
复制
发表时间：
1979-01-01
影响因子：
2.9
通讯作者：
NYGAARD, P
中科院分区：
文献类型：
--
作者：
JENSEN, KF;HOULBERG, U;NYGAARD, P
描述了用TLC方法测定5-磷酸核糖基-α-1-焦磷酸(PRPP)的条件，该方法通常用于测定~(32)P标记细菌中的核苷三磷酸。描述了一种二维层析系统，在该系统中可以测定非常小的PRPP池(约0.03微克/克干重细菌)。在一维层析系统中，检测PRPP池的下限约为0.3微克分子/克干重细菌。这个一维系统还提供了一种检测PRPP合成酶活性的方法，即使在使用[γ-32P]ATP作为底物的粗提物中也是如此。由于PRPP的层析分离，该方法具有高度的特异性；由于使用32P标记，该方法非常敏感。将该方法应用于鼠伤寒沙门氏菌的部分突变株的分析，结果与常规的分析结果吻合较好。
Conditions are described where 5-phosphoribosyl-.alpha.-1-pyrophosphate (PRPP) can be determined by TLC methods commonly used for the determination of nucleoside triphosphate pools in 32P-labeled bacteria. A 2-dimensional chromatographic system is described where very small pools of PRPP (about 0.03 .mu.mol/gram dry wt bacteria) can be determined. In a unidimensional chromatographic system the lower limit for detection of PRPP pools is about 0.3 .mu.mol/g dry wt bacteria. This unidimensional system offers an assay also for PRPP synthetase activity even in crude extracts using [.gamma.-32P]ATP as a substrate. The assay is highly specific due to the chromatographic isolation of PRPP and is very sensitive due to the use of 32P labeling. The chromatographic methods for determination of PRPP pools and of activities of PRPP synthetase were applied to the analysis of some mutants of Salmonella typhimurium and provided results that agree well with the results obtained by conventional methods of PRPP analysis.