Rosen Gerald M, Tsai Pei, Weaver John, Porasuphatana Supatra, Roman Linda J, Starkov Anatoly A, Fiskum Gary, Pou Sovitj
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 725 West Lombard Street, Baltimore, MD 21201, USA.
J Biol Chem. 2002 Oct 25;277(43):40275-80. doi: 10.1074/jbc.M200853200. Epub 2002 Aug 14.
Tetrahydrobiopterin (H(4)B) is a critical element in the nitric-oxide synthase (NOS) metabolism of l-arginine to l-citrulline and NO(.). It has been hypothesized that in the absence of or under nonsaturating levels of L-arginine where O(2) reduction is the primary outcome of NOS activation, H(4)B promotes the generation of H(2)O(2) at the expense of O(2)(-.). The experiments were designed to test this hypothesis. To test this theory, two different enzyme preparations, H(4)B-bound NOS I and H(4)B-free NOS I, were used. Initial rates of NADPH turnover and O(2) utilization were found to be considerably greater in the H(4)B-bound NOS I preparation than in the H(4)B-free NOS I preparation. In contrast, the initial generation of O(2)(-.) from the H(4)B-free NOS I preparation was found to be substantially greater than that measured using the H(4)B-bound NOS I preparation. Finally, by spin trapping nearly all of the NOS I produced O(2)(-.), we found that the initial rate of H(2)O(2) production by H(4)B-bound NOS I was considerably greater than that for H(4)B-free NOS I.
四氢生物蝶呤（H(4)B）是一氧化氮合酶（NOS）将L-精氨酸代谢为L-瓜氨酸和NO(.)过程中的关键要素。据推测，在缺乏L-精氨酸或L-精氨酸处于非饱和水平时，此时NOS激活的主要产物是氧的还原，H(4)B会以消耗超氧阴离子（O(2)(-.)）为代价促进过氧化氢（H(2)O(2)）的生成。设计这些实验旨在验证这一假说。为了验证该理论，使用了两种不同的酶制剂，即结合H(4)B的NOS I和未结合H(4)B的NOS I。结果发现，结合H(4)B的NOS I制剂中NADPH周转和氧利用的初始速率比未结合H(4)B的NOS I制剂中的要高得多。相反，发现未结合H(4)B的NOS I制剂中超氧阴离子（O(2)(-.)）的初始生成量明显高于使用结合H(4)B的NOS I制剂所测得的量。最后，通过自旋捕集几乎所有由NOS I产生的超氧阴离子（O(2)(-.)），我们发现结合H(4)B的NOS I产生过氧化氢（H(2)O(2)）的初始速率比未结合H(4)B的NOS I要高得多。
