Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides
Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides
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DOI：
10.1074/jbc.m312560200
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发表时间：
2004-05-07
影响因子：
4.8
通讯作者：
Seifert, R
中科院分区：
文献类型：
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作者：
Gille, A;Lushington, GH;Seifert, R
哺乳动物表达9种膜腺苷酸环化酶亚型（AC 1-9），一种结构相关的可溶性鸟苷酸环化酶（sGC）和一种可溶性AC（sAC）。此外，炭疽杆菌和百日咳杆菌分别产生AC毒素、水肿因子（EF）和腺苷酸环化酶毒素（ACT）。2 '（3'）-O-（N-methylanthraniloyl）guanosine 5 '-[gamma-thio]triphosphate是S49淋巴瘤细胞膜中AC的有效竞争性抑制剂。这些数据促使我们系统地研究了24个核苷酸对S49和Sf 9昆虫细胞膜中的AC、在Sf 9膜中表达的AC 1、2、5和6以及膜AC的纯化催化亚基（AC 5的C1和AC 2的C2）、sAC、sGC、EF和ACT在MnCl 2存在下的影响。N-甲基邻氨基苯甲酰（MANT）-GTP抑制C1.C2，Ki为4.2 nM。C2的Phe-889和Ile-940介导与MANT基团的疏水相互作用。MANT-肌苷5 '-[γ-硫代]三磷酸可有效抑制C1、C2和AC 1、5和6，但仅对sGC、EF、ACT和G蛋白表现出低亲和力。肌苷5 '-[γ-硫代]三磷酸和尿苷5'-[硫代]三磷酸是G蛋白激活剂和AC抑制剂的混合物。AC 5对抑制剂的敏感性是AC 2的15倍。EF和ACT表现出独特的抑制剂谱。在sAC下，2 '，5'-二脱氧腺苷3 '-三磷酸是最有效的化合物（IC 50，690 nM）。几种ANT-腺嘌呤和ANT-鸟嘌呤核苷酸抑制sGC，Ki值在200-400 nM范围内。UTP和ATP对sGC表现出与GTP相似的亲和力，并且是混合的sGC底物和抑制剂。MnCl 2与MgCl 2的交换使AC和sGC的抑制剂效力降低1.5-250倍，这取决于所研究的核苷酸和环化酶。从环化酶反应中省略NTP再生系统强烈降低了MANT-ADP的效力，表明丙酮酸激酶磷酸化为MANT-ATP。总的来说，AC亚型和sGC被嘌呤和嘧啶核苷酸差异性地抑制。
Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl2. N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K-i of 4.2 nM. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[thio] triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC50, 690 nM). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with Ki values in the 200-400 nM range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl2 against MgCl2 reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.