Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.
Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.
复制标题
颗粒鸟苷酸环化酶在大鼠肝脏质膜和微粒体中的定位。
DOI：
10.1016/s0021-9258(19)41376-8
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发表时间：
1975
期刊：
影响因子：
--
通讯作者：
F. Murad
中科院分区：
文献类型：
--
作者：
H. Kimura;F. Murad
研究了鸟苷酸环化酶在大鼠肝脏中的亚细胞定位。匀浆中约80%的酶活性存在于可溶性部分。颗粒鸟苷酸环化酶定位于质膜和微粒体。将粗核和微粒体组分应用于不连续的蔗糖梯度，并对所得组分进行鸟苷酸环化酶、细胞组分的各种酶标记物和电子显微镜检查。纯化后的质膜组分的鸟苷酸环化酶比活性最高，为30 ~ 80 pmol/min/mg蛋白质，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相似。在纯化制备的微粒体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。我们不能排除鸟苷酸环化酶存在于其他细胞成分中，如高尔基体。电镜观察结果支持酶标记的生化研究。可溶性鸟苷酸环化酶对GTP具有典型的Michaelis-Menten动力学，对GTP的表观Km为35mum。Ca-2+在低浓度Mn-2+的存在下刺激了可溶性活性。鸟苷酸环化酶在质膜和微粒体中的性质相似，只是Ca-2+抑制了与质膜相关的活性，而对微粒体的活性没有影响。两种颗粒酶本质上都是变构酶；速度与GTP的双倒数图不是线性关系，质膜和微粒体制备的Hill系数分别为1.60和1.58。可溶性酶和颗粒酶均受到ATP的抑制，其中可溶性酶的抑制作用略大。虽然Mg-2+作为唯一阳离子的效果不如Mn-2+，但在低浓度的Mn-2+存在下，Mg-2+对所有酶组分都有明显的刺激作用。Triton X-100将颗粒组分的活性提高了3- 10倍，将可溶性活性提高了50 - 100%。
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%.