A method for determination of pyridine nucleotides using a single extract
A method for determination of pyridine nucleotides using a single extract
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DOI：
10.1006/abio.2000.4701
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发表时间：
2000-10-01
影响因子：
2.9
通讯作者：
Stanton, RC
中科院分区：
文献类型：
--
作者：
Zhang, ZQ;Yu, J;Stanton, RC
细胞内氧化还原水平在生理学和病理生理学中起着重要作用。细胞内的主要还原剂是NADPH，它是整个抗氧化剂系统和重要的促氧化剂酶(如一氧化氮合酶和NADPH氧化酶)正常活动所必需的。因此，对NADPH进行简便、准确的测量是非常必要的。本文介绍的方法是基于NADH和NADPH(而不是NAD(+)和NADP(+))对340 nm处的吸光度产生影响的事实。单细胞提取物被分成三等份(A(1)、A(2)和A(3))。A(1)未经处理，测量了340 nm处的吸光度。A(2)用一种将所有NADP(+)转化为NADPH的酶处理，然后在340 nm处测量吸光度。A(2)用一种将所有NADPH转化为NADP(+)的酶处理，然后在340 nm处测量吸光度。A(1)-A(3)是提取物的NADPH含量，A(2)-A(1)是提取物的NADP(+)含量。用该方法从细胞提取液中完全回收了添加的核苷酸，适用于活细胞中NADP(+)和NADPH的快速测定。结论该方法测定NADP(+)和NADPH具有快速、简便、准确、可靠的特点。(C)2000年学术出版社。
Intracellular redox levels play an important role in physiology and pathophysiology. The principal intracellular reductant is NADPH, which is required for both the proper activity of the entire antioxidant system and important prooxidant enzymes such as nitric oxide synthase and NADPH oxidase. Thus an easy and accurate measurement of NADPH is very desirable. The method described in this paper is based on the fact that NADH and NADPH (not NAD(+) and NADP(+)) affect absorbance at 340 nm. A single cell extract is separated into three aliquots (A(1), A(2), and A(3)). A(1) is untreated and the absorbance at 340 nm is measured. A(2) is treated with an enzyme that converts all of the NADP(+) to NADPH and then the absorbance at 340 nm is measured. A(2) is treated with an enzyme that converts all of the NADPH to NADP(+) and then the absorbance at 340 nm is measured. A(1) - A(3) is the NADPH content and A(2) - A(1) is the NADP(+) content of the extract. Using this method, we have obtained full recovery of all added nucleotides from cell extracts, thus making the method suitable for the quick determination of NADP(+) and NADPH in living cells. We conclude that this method for the measurement of NADP(+) and NADPH is rapid, simple, accurate, and reliable. (C) 2000 Academic Press.