Deguchi T
J Biol Chem. 1977 Jan 25;252(2):596-601.
Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.
羟胺激活大鼠大脑皮质微粒体部分的鸟苷酸环化酶。在粗线粒体部分激活最为显著。当粗线粒体部分受到渗透压休克并分级分离时，用羟胺测定，亚组分中恢复的鸟苷酸环化酶活性仅为起始材料的三分之一。然而，可溶性部分和微粒体部分的重组将鸟苷酸环化酶活性恢复到与起始材料相同的水平。当将不同量的微粒体部分和可溶性部分混合时，酶活性与可溶性部分的量成正比。将可溶性部分或微粒体部分在55℃加热5分钟会使鸟苷酸环化酶失活。加热的微粒体部分显著激活天然可溶性部分中的鸟苷酸环化酶活性，而加热的可溶性部分不会刺激微粒体中的酶活性。将微粒体部分在37℃与羟胺预孵育5分钟，然后洗涤，在没有羟胺的情况下激活了可溶性部分中的鸟苷酸环化酶活性。对粗线粒体部分的进一步分级分离表明，羟胺激活所需的因子与线粒体相关。大脑皮质的线粒体部分在羟胺存在下激活脑、肝或肾上清液中的鸟苷酸环化酶。反过来，从肝或肾制备的线粒体部分激活脑中的可溶性鸟苷酸环化酶。将羟胺对鸟苷酸环化酶的激活作用与叠氮化钠的激活作用进行了比较。叠氮化物激活突触体可溶性部分中的鸟苷酸环化酶，而羟胺则抑制它。在没有叠氮化物的情况下，预先用叠氮化物孵育然后洗涤的微粒体部分不会刺激鸟苷酸环化酶活性。羟胺对鸟苷酸环化酶的激活作用不是由于底物GTP浓度的变化，添加羟胺不会改变鸟苷酸环化酶对GTP的表观Km值。在羟胺存在下，鸟苷酸环化酶对锰的依赖性降低。因此，羟胺对鸟苷酸环化酶的激活作用是由于反应的Vmax发生了变化。
