King M T, Passonneau J V, Veech R L
National Institute on Alcohol Abuse and Alcoholism, Laboratory of Metabolism and Molecular Biology, Rockville, Maryland 20852.
Anal Biochem. 1990 May 15;187(1):179-86. doi: 10.1016/0003-2697(90)90438-f.
Methods for the measurement of phosphoribosylpyrophosphate (PRPP) and ribose 5-phosphate (R-5-P) in tissues have been developed. The lability of these compounds during tissue extraction and the recovery of standards from tissue preparations have been examined. Enzymatic conversion of phosphoribosylpyrophosphate to [14C]AMP in the presence of labeled adenine or formation of [14C]GMP ([14C]IMP) in the presence of labeled guanine or hypoxanthine was accomplished in the first step. In the second step, the labeled product was separated from the substrate. For the measurement of R-5-P, the first step included phosphoribosylpyrophosphate synthetase, as well as the appropriate substrate and effector (ATP and Pi), in combination with adenine phosphoribosyl transferase. The product [14C]AMP was measured in three ways: (1) HPLC separation with an on-line radioisotope detector; (2) butanol extraction of the labeled base, and measurement of an aliquot of the aqueous phase in a scintillation counter; (3) filtration of the incubation mixture with chromatographic filter paper disks, which were then counted in a scintillation counter. When [14C]guanine was the substrate, HPLC separation was used because the butanol or paper separation was not adequate. Measurement of 5-125 pmol of PRPP or R-5-P gave a linear response.
已开发出用于测量组织中磷酸核糖焦磷酸（PRPP）和5-磷酸核糖（R-5-P）的方法。已研究了这些化合物在组织提取过程中的稳定性以及从组织制剂中回收标准品的情况。第一步是在标记的腺嘌呤存在下将磷酸核糖焦磷酸酶促转化为[14C]AMP，或在标记的鸟嘌呤或次黄嘌呤存在下形成[14C]GMP（[14C]IMP）。第二步，将标记产物与底物分离。对于R-5-P的测量，第一步包括磷酸核糖焦磷酸合成酶以及合适的底物和效应物（ATP和Pi），并结合腺嘌呤磷酸核糖转移酶。产物[14C]AMP通过三种方式进行测量：（1）使用在线放射性同位素检测器进行HPLC分离；（2）用丁醇萃取标记的碱基，并在闪烁计数器中测量水相的一份样品；（3）用色谱滤纸圆盘过滤孵育混合物，然后在闪烁计数器中计数。当以[14C]鸟嘌呤为底物时，使用HPLC分离，因为丁醇或纸分离方法不适用。测量5-125 pmol的PRPP或R-5-P可得到线性响应。
