STUDIES OF THE FERRICYANIDE REDUCTASE ACTIVITIES OF THE MITOCHONDRIAL REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE-UBIQUINONE REDUCTASE (COMPLEX I) UTILIZING ARYLAZIDO-BETA-ALANYL NAD+ AND ARYLAZIDO-BETA-ALANYL NADP+
STUDIES OF THE FERRICYANIDE REDUCTASE ACTIVITIES OF THE MITOCHONDRIAL REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE-UBIQUINONE REDUCTASE (COMPLEX I) UTILIZING ARYLAZIDO-BETA-ALANYL NAD+ AND ARYLAZIDO-BETA-ALANYL NADP+
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DOI：
10.1007/bf00744987
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发表时间：
1985-01-01
影响因子：
3
通讯作者：
GUILLORY, RJ
中科院分区：
文献类型：
--
作者：
CHEN, S;GUILLORY, RJ
利用2种光亲和性吡啶核苷酸类似物：芳基叠氮基-β-吡啶基-β-吡啶基，研究了线粒体NADH-CoQ还原酶制剂中存在的NADH和NADPH铁氰化物还原酶活性。丙氨酰NAD+（A3“-O-{3-[N-（4-叠氮基-2-硝基苯基）氨基]丙酰基}NAD+）和芳基叠氮基-β-丙氨酰NADP+（N3“-O-{3-[N-（4-叠氮基-3-硝基苯基）氨基]-丙酰基}NADP+）。对于NADH-K3 Fe（CN）6还原酶活性，芳基叠氮基-β-发现在黑暗中丙氨酰NAD+是相对于NADH和K3 Fe（CN）6的竞争性抑制剂，Ki，app [表观Ki]值分别为9.7和15.5 μ M。相比之下，NADP+类似物对这两种底物的反应表现出弱的非竞争性抑制剂活性。在光照射下，芳基叠氮基-β-在超过酶浓度25倍摩尔过量的类似物存在下，丙氨酰NAD+抑制NADH-K3 Fe（CN）6还原酶高达70%。这种光依赖性抑制可以防止的存在下，在照射过程中，天然底物NADH。与此相反，复杂的动力学结果，获得了与吡啶核苷酸类似物的NADPH-K3 Fe（CN）6还原酶活性的影响，在黑暗中的研究。在酶复合物的存在下，任何一种类似物的光照射导致NADPH依赖性活性的激活。复合物I的NADPH-K_3Fe（CN）_6还原酶活性可能是NADPH-NAD+转氢酶和NADH氧化还原酶的铁氰化物还原酶活性的总和。
The NADH and NADPH ferricyanide reductase activities present in mitochondrial NADH-CoQ reductase preparations were studied utilizing 2 photoaffinity pyridine nucleotide analogs: arylazido-.beta.-alanyl NAD+ (A3''-O-{3-[N-(4-azido-2-nitrophenyl)amino]propionyl}NAD+) and arylazido-.beta.-alanyl NADP+ (N3''-O-{3-[N-(4-azido-3-nitrophenyl)amino]-propionyl}NADP+). For the NADH-K3Fe(CN)6 reductase activity, arylazido-.beta.-alanyl NAD+ was found to be, in the dark, a competitive inhibitor with respect to both NADH and K3Fe(CN)6 with Ki,app [apparent Ki] values of 9.7 and 15.5 .mu.M, respectively. In comparison the NADP+ analog exhibited weak noncompetitive inhibitor activity for this reaction against both substrates. Upon photoirradiation arylazido-.beta.-alanyl NAD+ inhibited NADH-K3Fe(CN)6 reductase up to 70% in the presence of a 25-fold molar excess of analog over the enzyme concentration. This photodependent inhibition could be prevented by the presence, during irradiation, of the natural substrate NADH. In contrast complex kinetic results were obtained with studies of the effects of the pyridine nucleotide analogues of NADPH-K3Fe(CN)6 reductase activity in the dark. Photoirradiation of either analog in the presence of the enzyme complex resulted in an activation of NADPH-dependent activity. The possibility that the NADPH-K3Fe(CN)6 reductase activity of complex I represents a summation of the combined ferricyanide reductase activity of the NADPH-NAD+ transhydrogenase and NADH oxidoreductase is suggested.