Kimura H, Murad F
J Biol Chem. 1975 Jun 25;250(12):4810-7.
The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.
对大鼠肝脏中的鸟苷酸环化酶进行了亚细胞定位研究。匀浆中约80%的酶活性存在于可溶性部分。颗粒状鸟苷酸环化酶定位于质膜和微粒体中。将粗制的细胞核和微粒体部分应用于不连续蔗糖梯度，对所得部分进行鸟苷酸环化酶、细胞成分的各种酶标志物以及电子显微镜检查。从两种制备物中获得的纯化质膜部分具有最高的鸟苷酸环化酶比活性，为30至80 pmol/分钟/毫克蛋白质，并且这些部分中鸟苷酸环化酶的回收率和相对比活性与5'-核苷酸酶和腺苷酸环化酶的回收率和相对比活性平行。在纯化的微粒体制备物中回收了大量的鸟苷酸环化酶、腺苷酸环化酶、5'-核苷酸酶和葡萄糖-6-磷酸酶。我们不能排除其他细胞成分如高尔基体中存在鸟苷酸环化酶。各部分的电子显微镜研究支持了用酶标志物进行的生化研究。可溶性鸟苷酸环化酶对GTP具有典型的米氏动力学，其对GTP的表观Km为35μM。在低浓度Mn-2+存在的情况下，Ca-2+刺激可溶性活性。质膜和微粒体中鸟苷酸环化酶的性质相似，只是Ca-2+抑制与质膜相关的活性，而对微粒体的活性没有影响。两种颗粒状酶本质上都是别构的；速度与GTP的双倒数图不是线性的，质膜和微粒体制备物的希尔系数分别计算为1.60和1.58。可溶性和颗粒状酶均受到ATP的抑制，对可溶性酶的抑制作用稍大。虽然Mg-2+作为唯一阳离子的效果不如Mn-2+，但在低浓度Mn-2+存在的情况下，所有酶部分都受到Mg-2+的显著刺激。Triton X-100使颗粒状部分的活性增加约3至10倍，使可溶性活性增加50%至100%。
