Li J, Pak J H, Huang F L, Huang K P
Section on Metabolic Regulation, Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
J Biol Chem. 1999 Jan 15;274(3):1294-300. doi: 10.1074/jbc.274.3.1294.
Neurogranin/RC3 (Ng), a postsynaptic neuronal protein kinase C (PKC) substrate, binds calmodulin (CaM) at low level of Ca2+. In vitro, rat brain Ng can be oxidized by nitric oxide (NO) donors and by oxidants to form an intramolecular disulfide bond with resulting downward mobility shift on nonreducing SDS-polyacrylamide gel electrophoresis. The oxidized Ng, as compared with the reduced form, is a poorer substrate of PKC but like the PKC-phosphorylated Ng has a lower affinity for CaM than the reduced form. To investigate the physiological relevance of Ng oxidation, we tested the effects of neurotransmitter, N-methyl-D-aspartate (NMDA), NO donors, and other oxidants such as hydrogen peroxide and oxidized glutathione on the oxidation of this protein in rat brain slices. Western blot analysis showed that the NMDA-induced oxidation of Ng was rapid and transient, it reached maximum within 3-5 min and declined to base line in 30 min. The response was dose-dependent (EC50 approximately 100 microM) and could be blocked by NMDA-receptor antagonist 2-amino-5-phosphonovaleric acid and by NO synthase inhibitor NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine. Ng was oxidized by NO donors, sodium nitroprusside, S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione, and H2O2 at concentrations less than 0.5 mM. Oxidation of Ng in brain slices induced by sodium nitroprusside could be reversed by dithiothreitol, ascorbic acid, or reduced glutathione. Reversible oxidation and reduction of Ng were also observed in rat brain extracts, in which oxidation was enhanced by Ca2+ and the oxidized Ng could be reduced by NADPH or reduced glutathione. These results suggest that redox of Ng is involved in the NMDA-mediated signaling pathway and that there are enzymes catalyzing the oxidation and reduction of Ng in the brain. We speculate that the redox state of Ng, similar to the state of phosphorylation of this protein, may regulate the level of CaM, which in turn modulates the activities of CaM-dependent enzymes in the neurons.
神经颗粒素/RC3（Ng）是一种突触后神经元蛋白激酶C（PKC）底物，在低钙离子水平时与钙调蛋白（CaM）结合。在体外，大鼠脑Ng可被一氧化氮（NO）供体和氧化剂氧化，形成分子内二硫键，在非还原SDS-聚丙烯酰胺凝胶电泳上导致迁移率下降。与还原形式相比，氧化后的Ng是PKC较差的底物，但与PKC磷酸化的Ng一样，对CaM的亲和力低于还原形式。为了研究Ng氧化的生理相关性，我们测试了神经递质N-甲基-D-天冬氨酸（NMDA）、NO供体以及其他氧化剂如过氧化氢和氧化型谷胱甘肽对大鼠脑片该蛋白氧化的影响。蛋白质印迹分析表明，NMDA诱导的Ng氧化迅速且短暂，在3 - 5分钟内达到最大值，并在30分钟内降至基线。该反应呈剂量依赖性（半数有效浓度约为100微摩尔），可被NMDA受体拮抗剂2-氨基-5-磷酸戊酸以及NO合酶抑制剂NG-硝基-L-精氨酸甲酯和NG-单甲基-L-精氨酸阻断。Ng可被NO供体硝普钠、S-亚硝基-N-乙酰青霉胺和S-亚硝基谷胱甘肽以及浓度低于0.5毫摩尔的过氧化氢氧化。硝普钠诱导的脑片Ng氧化可被二硫苏糖醇、抗坏血酸或还原型谷胱甘肽逆转。在大鼠脑提取物中也观察到了Ng的可逆氧化和还原，其中氧化作用被钙离子增强，氧化后的Ng可被NADPH或还原型谷胱甘肽还原。这些结果表明，Ng的氧化还原参与了NMDA介导的信号通路，并且大脑中存在催化Ng氧化和还原的酶。我们推测，Ng的氧化还原状态与该蛋白的磷酸化状态类似，可能调节CaM的水平，进而调节神经元中CaM依赖性酶的活性。
