Kamisaki Y, Waldman S A, Murad F
Arch Biochem Biophys. 1986 Dec;251(2):709-14. doi: 10.1016/0003-9861(86)90380-2.
Sodium nitroprusside, a potent activator of soluble guanylate cyclase, potentiated mixed disulfide formation between cystine, a potent inhibitor of the cyclase, and enzyme purified from rat lung. Incubation of soluble guanylate cyclase with nitroprusside and [35S]cystine resulted in a twofold increase in protein-bound radioactivity compared to incubations in the absence of nitroprusside. Purified enzyme preincubated with nitroprusside and then gel filtered (activated enzyme) was activated 10- to 20-fold compared to guanylate cyclase preincubated in the absence of nitroprusside and similarly processed (nonactivated enzyme). This activation was completely reversed by subsequent incubation at 37 degrees C (activation-reversed enzyme). Incorporation of [35S]cystine into guanylate cyclase was increased twofold with activated enzyme, while no difference was observed with activation-reversed enzyme, compared to nonactivated enzyme. Cystine decreased the activity of nonactivated and activation-reversed enzyme about 40% while it completely inhibited activated guanylate cyclase. Mg+2- or Mn+2-GTP inhibited the incorporation of [35S]cystine into nonactivated or activated guanylate cyclase. Also, diamide, a potent thiol oxidant that converts juxtaposed sulfhydryls to disulfides, completely blocked incorporation of [35S]cystine into nonactivated or activated guanylate cyclase. These data indicate that activation of soluble guanylate cyclase by nitroprusside results in an increased availability of protein sulfhydryl groups for mixed disulfide formation with cystine. Protection against mixed disulfide formation with diamide or substrate suggests that these groups exist as two or more juxtaposed sulfhydryl groups at the active site or a site on the enzyme that regulates catalytic activity. Differential inhibition by mixed disulfide formation of nonactivated and activated enzyme suggests a mechanism for amplification of the on-off signal for soluble guanylate cyclase within cells.
硝普钠是可溶性鸟苷酸环化酶的强效激活剂，它能增强胱氨酸（该环化酶的强效抑制剂）与从大鼠肺中纯化得到的酶之间混合二硫键的形成。将可溶性鸟苷酸环化酶与硝普钠和[35S]胱氨酸一起孵育，与在无硝普钠条件下孵育相比，蛋白质结合的放射性增加了两倍。预先用硝普钠孵育然后进行凝胶过滤的纯化酶（活化酶），与在无硝普钠条件下预先孵育并进行类似处理的鸟苷酸环化酶（未活化酶）相比，活性提高了10至20倍。在37℃下后续孵育可使这种活化完全逆转（活化逆转酶）。与未活化酶相比，活化酶使[35S]胱氨酸掺入鸟苷酸环化酶的量增加了两倍，而活化逆转酶则未观察到差异。胱氨酸使未活化酶和活化逆转酶的活性降低约40%，而它能完全抑制活化的鸟苷酸环化酶。Mg+2-或Mn+2-GTP抑制[35S]胱氨酸掺入未活化或活化的鸟苷酸环化酶。此外，二酰胺是一种强效的硫醇氧化剂，可将相邻的巯基转化为二硫键，它完全阻断了[35S]胱氨酸掺入未活化或活化的鸟苷酸环化酶。这些数据表明，硝普钠对可溶性鸟苷酸环化酶的激活导致蛋白质巯基与胱氨酸形成混合二硫键的可用性增加。用二酰胺或底物防止混合二硫键形成表明，这些基团在活性位点或调节催化活性的酶位点上以两个或更多相邻的巯基形式存在。未活化酶和活化酶因混合二硫键形成而产生的差异抑制表明了一种细胞内可溶性鸟苷酸环化酶开-关信号放大的机制。
