Modification of cardiac membrane adenylate cyclase activity and Gs alpha by NAD and endogenous ADP-ribosyltransferase.
Modification of cardiac membrane adenylate cyclase activity and Gs alpha by NAD and endogenous ADP-ribosyltransferase.
复制标题
NAD 和内源 ADP-核糖基转移酶对心脏膜腺苷酸环化酶活性和 Gs α 的修饰。
DOI：
10.1006/jmcc.1994.1028
复制
发表时间：
1994
影响因子：
5
通讯作者：
Jacobson,MK
中科院分区：
文献类型：
--
作者：
Quist,EE;Coyle,DL;Vasan,R;Satumtira,N;Jacobson,EL;Jacobson,MK
探讨了NAD激活心肌腺苷环化酶的机制。在高纯度犬心肌肌膜上，NAD可在GS激动剂(即5 mM AlF4，10μM GTPγS.10μM GppNHp或异丙肾上腺素+2 NM GTPγS)存在下，刺激腺苷环化酶的活性。此外，NAD还降低了异丙肾上腺素对腺苷环化酶的EC50。在与[~(32)P]-NAD、AlF_4~-、10μ~(-1)GTPγS或异丙肾上腺素+2 NM~(-1)GTPγS共同孵育的膜上，经免疫沉淀鉴定为GS-α的45-KDA蛋白的放射性标记选择性增加。霍乱毒素催化了同一蛋白质的放射性标记。中性羟胺在AlF4-和[32P]-NAD存在下从预先标记的GSα中释放出[32P]-腺苷二磷酸核糖，表明GSα上的精氨酸残基被内源性的腺苷二磷酸核糖转移酶修饰。腺苷二磷酸核糖转移酶抑制剂、新诺维菌素、维生素K1或3-氨基苯甲酰胺均能抑制AlF4刺激的GSα的腺苷二磷酸核糖化和腺苷环化酶的NAD增强。在低渗或低渗条件下，负责腺苷环化酶和GSα腺苷二磷酸核糖化的活性没有被去除，因此似乎是紧密结合在膜上的。综上所述，这些观察结果表明，犬心肌肌膜上具有腺苷二磷酸核糖转移酶，该酶可结构性地催化腺苷二磷酸核糖转移到激活的GSα。
The mechanism by which NAD stimulates cardiac adenylate cyclase was investigated. In highly purified canine cardiac sarcolemma, NAD stimulated adenylate cyclase activity in the presence of agents which activate GS(i.e. 5 mM AlF4-, 10 μM GTPγS. 10 μM GppNHp or isoproterenol plus 2 nM GTPγS). Furthermore the EC50of isoproterenol of stimulate adenylate cyclase was reduced in the presence of NAD. In membranes incubated with [32P]-NAD, AlF4-, 10 μM GTPγS or isoproterenol plus 2 nM GTPγS produced a selective increase in the radiolabeling of a single 45-kDA protein which was identified as GSαby immunoprecipitation. Cholera toxin catalysed radiolabeling of the same protein. Neutral hydroxylamine released [32P]-ADP-ribose from GSαprelabeled in the presence of AlF4-and [32P]-NAD indicating that an arginine residue on GSαwas modified by an endogenous ADP-ribosyltransferase. ADP-ribosyltransferase inhibitors, novobiocin, vitamin K1or 3-aminobenzamide, inhibited AlF4-stimulated ADP-ribosylation of GSαand NAD potentiation of adenylate cyclase with similar efficacies. The activity responsible for NAD potentiation of adenylate cyclase and ADP-ribosylation of GSαwas not removed under hypotonic or hypotonic conditions and therefore appears to be tightly membrane bound. Collectively, these observations indicate that canine cardiac sarcolemma possess and ADP-ribosyltransferase which may constitutively catalyse transfer of an ADP-ribose to activated GSα.