Kimura H, Mittal C K, Murad F
J Biol Chem. 1975 Oct 25;250(20):8016-22.
Sodium azide, hydroxylamine, and phenylhydrazine at concentrations of 1 mM increased the activity of soluble guanylate cyclase from rat liver 2- to 20-fold. The increased accumulation of guanosine 3':5'-monophosphate in reaction mixtures with sodium azide was not due to altered levels of substrate, GTP, or altered hydrolysis of guanosine 3':5'-monophosphate by cyclic nucleotide phosphodiesterase. The activation of guanylate cyclase was dependent upon NaN3 concentration and temperature; preincubation prevented the time lag of activation observed during incubation. The concentration of NaN3 that resulted in half-maximal activation was 0.04 mM. Sodium azide increased the apparent Km for GTP from 35 to 113 muM. With NaN3 activation the enzyme was less dependent upon the concentration of free Mn2+. Activation of enzyme by NaN3 was irreversible with dilution or dialysis of reaction mixtures. The slopes of Arrhenius plots were altered with sodium azide-activated enzyme, while gel filtration of the enzyme on Sepharose 4B was unaltered by NaN3 treatment. Triton X-100 increased the activity of the enzyme, and in the presence of Triton X-100 the activation by NaN3 was not observed. Trypsin treatment decreased both basal guanylate cyclase activity and the responsiveness to NaN3. Phospholipase A, phospholipase C, and neuraminidase increased basal activity but had little effect on the responsiveness to NaN3. Both soluble and particulate guanylate cyclase from liver and kidney were stimulated with NaN3. The particulate enzyme from cerebral cortex and cerebellum was also activated with NaN3, whereas the soluble enzyme from these tissues was not. Little or no effect of NaN3 was observed with preparations from lung, heart, and several other tissues. The lack of an effect with NaN3 on soluble GUANYLATE Cyclase from heart was probably due to the presence of an inhibitor of NaN3 activation in heart preparations. The effect of NaN3 was decreased or absent when soluble guanylate cyclase from liver was purified or stored at -20degrees. The activation of guanylate cyclase by NaN3 is complex and may be the result of the nucleophilic agent acting on the enzyme directly or what may be more likely on some other factor in liver preparations.
浓度为1 mM的叠氮化钠、羟胺和苯肼可使大鼠肝脏可溶性鸟苷酸环化酶的活性提高2至20倍。在含有叠氮化钠的反应混合物中，3':5'-环磷酸鸟苷积累的增加并非由于底物GTP水平的改变，也不是由于环核苷酸磷酸二酯酶对3':5'-环磷酸鸟苷水解的改变。鸟苷酸环化酶的激活取决于叠氮化钠的浓度和温度；预孵育可防止孵育期间观察到的激活延迟。导致半数最大激活的叠氮化钠浓度为0.04 mM。叠氮化钠使GTP的表观Km从35 μM增加到113 μM。在叠氮化钠激活的情况下，该酶对游离Mn2+浓度的依赖性降低。反应混合物经稀释或透析后，叠氮化钠对酶的激活作用不可逆。叠氮化钠激活的酶的阿累尼乌斯图斜率发生改变，而用叠氮化钠处理后，该酶在琼脂糖4B上的凝胶过滤未发生改变。Triton X-100可提高该酶的活性，且在Triton X-100存在的情况下未观察到叠氮化钠的激活作用。胰蛋白酶处理可降低鸟苷酸环化酶的基础活性以及对叠氮化钠的反应性。磷脂酶A、磷脂酶C和神经氨酸酶可提高基础活性，但对叠氮化钠反应性的影响很小。肝脏和肾脏的可溶性和颗粒性鸟苷酸环化酶均受到叠氮化钠的刺激。大脑皮层和小脑的颗粒性酶也被叠氮化钠激活，而这些组织的可溶性酶则未被激活。在肺、心脏和其他几种组织的制剂中，未观察到叠氮化钠有明显作用。叠氮化钠对心脏可溶性鸟苷酸环化酶无作用可能是由于心脏制剂中存在叠氮化钠激活的抑制剂。当肝脏可溶性鸟苷酸环化酶被纯化或在-20℃储存时，叠氮化钠的作用减弱或消失。叠氮化钠对鸟苷酸环化酶的激活作用很复杂，可能是亲核试剂直接作用于该酶的结果，或者更有可能是作用于肝脏制剂中的某些其他因素。
