Chalmers A H
Aust J Exp Biol Med Sci. 1984 Jun;62 ( Pt 3):281-90. doi: 10.1038/icb.1984.28.
A simplified method for the estimation of 5-phosphoribosyl 1-pyrophosphate (PRPP) in mouse liver and spleen is described. The method uses the enzymic conversion of [8-14C] hypoxanthine to [8-14C] inosine 5'-monophosphate in the presence of PRPP and has the advantage over previously published methods in that the enzyme used in this assay, hypoxanthine-guanine phosphoribosyltransferase (HG-PRTase), is present in the tissue being analyzed and does not require preparation. In addition, each assay uses as its internal standard PRPP, thus overcoming the problem of the chemical instability inherent in this molecule. The two-minute heat treatment used in the method was found to destroy most interfering enzymes and resulted in average recovery of PRPP of 56% and 85% for the liver and spleen, respectively. The counts obtained in the assay were about ten-fold above background (80 c.p.m) and allowed accurate measurement of the phosphoribose sugar. The levels of PRPP (+/- standard deviation) found for liver and spleen in twelve (12) separate Balb/C male mice were 6.3 +/- 1.8 and 10.8 +/- 5.0 nmoles/g wet weight tissue, respectively, and agree with the tissue concentrations already reported for this metabolite. A further advantage of this assay is that it gives an index of the HG-PRTase activity in the tissues.
本文描述了一种用于估算小鼠肝脏和脾脏中5-磷酸核糖-1-焦磷酸（PRPP）的简化方法。该方法利用PRPP存在时，[8-¹⁴C]次黄嘌呤向[8-¹⁴C]肌苷5'-单磷酸的酶促转化，与先前发表的方法相比，其优势在于该测定中使用的酶——次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HG-PRTase）存在于被分析的组织中，无需制备。此外，每次测定都以PRPP作为内标，从而克服了该分子固有的化学不稳定性问题。该方法中使用的两分钟热处理被发现可破坏大多数干扰酶，肝脏和脾脏中PRPP的平均回收率分别为56%和85%。测定中获得的计数比背景值（80计数/分钟）高约十倍，能够准确测量磷酸核糖糖。在十二只（12只）单独的Balb/C雄性小鼠中，肝脏和脾脏中PRPP的水平（±标准差）分别为6.3±1.8和10.8±5.0纳摩尔/克湿重组织，与该代谢物已报道的组织浓度一致。该测定的另一个优点是它给出了组织中HG-PRTase活性的指标。
