White A A, Crawford K M, Patt C S, Lad P J
J Biol Chem. 1976 Dec 10;251(23):7304-12.
A 37,000 X g supernatant fraction prepared from fat lung homogenate demonstrated a 2- to 3-fold increase in guanylate cyclase activity after incubation at 30 degrees for 30 min (preincubation). Treatment of the supernatant fraction with Triton X-100 increased activity to approximately the same extent as preincubation, but would not increase the activity after preincubation. By chromatography on Sepharose 2B, before and after preincubation, it was demonstrated that the increase in activity was only associated with the soluble guanylate cyclase, and not the particulate enzyme. Activation by preincubation required O2. It was completely inhibited by thiols such as 2-mercaptoethanol, and by bovine serum albumin, KCN, and sodium diethyldithiocarbamate. These inhibitors suggested a copper requirement for activation, and this was confirmed by demonstrating that 20 to 60 muM CuCl2 could relieve the inhibition by 0.1 mM sodium diethyldithiocarbamate. 2-Mercaptoethanol inhibition could also be reversed by removal of the thiol on a Sephadex G-25 column, however, this treatment partially activated the enzyme. Addition of 2-mercaptoethanol to a preincubated preparation would not reverse the activation. H2O2 was found to activate guanylate cyclase, either by its generation in the lung supernatant with glucose oxidase and glucose, or by its addition to a preparation in which the catalase was inhibited with KCN. KCN or bovine serum albumin was able to partially inhibit activation by glucose oxidase plus glucose, however, larger amounts of glucose oxidase could overcome that inhibition, indicating a catalytic role for Cu2+ at low H2O2 concentrations. No direct evidence for H2O2 formation during preincubation could be found, however, indirect evidence was obtained by the spectrophotometric detection of choleglobin formation from hemoglobin present in the lung supernatant fluid. The H2O2 is believed to result from the reaction of oxyhemoglobin with ascorbate.
从脂肪肝匀浆制备的37,000 X g上清液组分，在30℃孵育30分钟（预孵育）后，鸟苷酸环化酶活性增加了2至3倍。用Triton X - 100处理上清液组分，活性增加的程度与预孵育大致相同，但预孵育后再用Triton X - 100处理则不会增加活性。通过在预孵育前后用Sepharose 2B进行层析，结果表明活性增加仅与可溶性鸟苷酸环化酶有关，而与颗粒酶无关。预孵育激活需要氧气。它被诸如2 - 巯基乙醇的硫醇、牛血清白蛋白、KCN和二乙基二硫代氨基甲酸钠完全抑制。这些抑制剂表明激活需要铜，这一点通过证明20至60 μM的CuCl₂可以解除0.1 mM二乙基二硫代氨基甲酸钠的抑制作用得到证实。2 - 巯基乙醇的抑制作用也可以通过在Sephadex G - 25柱上去除硫醇来逆转，然而，这种处理会使酶部分激活。向预孵育的制剂中添加2 - 巯基乙醇不会逆转激活作用。发现H₂O₂可激活鸟苷酸环化酶，要么通过在肺上清液中与葡萄糖氧化酶和葡萄糖一起产生H₂O₂，要么通过将其添加到用KCN抑制过氧化氢酶的制剂中。KCN或牛血清白蛋白能够部分抑制葡萄糖氧化酶加葡萄糖的激活作用，然而，大量的葡萄糖氧化酶可以克服这种抑制作用，表明在低H₂O₂浓度下Cu²⁺具有催化作用。在预孵育期间未发现H₂O₂形成的直接证据，然而，通过分光光度法检测肺上清液中存在的血红蛋白形成胆绿蛋白获得了间接证据。据信H₂O₂是由氧合血红蛋白与抗坏血酸反应产生的。
