通过放射免疫分析法对来自同一酸提取物中皮摩尔量的氧化型和还原型NAD及NADP的总量进行测定。

Determination by radioimmunoassay of the sum of oxidized and reduced forms of NAD and NADP in picomole quantities from the same acid extract.

作者信息

Bredehorst R, Lengyel H, Hilz H

出版信息

Eur J Biochem. 1979 Sep;99(2):401-11. doi: 10.1111/j.1432-1033.1979.tb13269.x.

DOI:10.1111/j.1432-1033.1979.tb13269.x

Abstract

The sum of the amounts of NAD + NADH was determined from the same acid tissue extract with the aid of a highly specific radioimmunoassay for 5'-AMP. NAD was converted to 5'-AMP via ADP-ribose by alkaline treatment while NADH was converted first to ADP-ribose by incubation of the acid extract at 25 degrees C followed by alkaline conversion to 5'-AMP. Removal of phosphate groups in NADP and NADPH by treatment of the extracts with alkaline phosphatase extended the procedure to the quantification of NADP(H). When combined with enzymic analyses of the oxidized coenzyme forms, NAD/NADH and NADP/NADPH ratios could also be obtained from the same extracts. The sensitivity of the test allows quantification of pyridine nucleotides in the range of 0.1--10 pmol.

摘要

借助针对5'-AMP的高特异性放射免疫分析法，从相同的酸性组织提取物中测定NAD + NADH的总量。通过碱性处理，NAD经由ADP-核糖转化为5'-AMP，而NADH则先通过在25℃下孵育酸性提取物转化为ADP-核糖，随后经碱性处理转化为5'-AMP。用碱性磷酸酶处理提取物以去除NADP和NADPH中的磷酸基团，从而将该方法扩展至对NADP(H)的定量分析。当与氧化型辅酶形式的酶促分析相结合时，还可从相同提取物中获得NAD/NADH和NADP/NADPH比率。该检测方法的灵敏度允许对0.1 - 10皮摩尔范围内的吡啶核苷酸进行定量分析。