Cheng H, Farquhar M G
J Cell Biol. 1976 Sep;70(3):660-70. doi: 10.1083/jcb.70.3.660.
The distribution of adenylate cyclase (AC) in Golgi and other cell fractions from rat liver was studied using the Golgi isolation procedure of Ehrenreich et al. In liver homogenate the AC activity was found to decay with time, but addition of 1 mM EGTA reduced the rate of enzyme loss. The incorporation of 1 mM EGTA into the sucrose medium used in the initial two centrifugal steps of the Golgi isolation method stabilized the enzyme activity throughout the entire procedure and resulted in good enzyme recovery. In such preparations, AC activity was demonstrated to be associated not only with plasma membranes but also with Golgi membranes and smooth microsomal membranes as well. Furthermore, under the conditions used, enzyme activity was also associated with the 105,000 g x 90 min supernatant fraction. The specific activity of the liver homogenate was found to be 2.9 pmol-mg protein-1-min-1, the nonsedimentabel and microsomal activity was of the same order of magnitude, but the Golgi and plasma membrane activities were much higher. The specific activity of plasma membrane AC was 29 pmol-mg proten-1-min-1. The Golgi activity varied in the three fractions, with the highest activity (14 pmol) in GF1 lowest activity (1.8) in GF2, and intermediate activity (5.5) in GF3, when the Golgi activity was corrected for the presence of content protein, the activity in GF1 became much higher (9 x) than that of the plasma membrane while the activities in GF2 and GF3 were comparable to that of plasma membrane. In all locations studied, the AC was sensitive to NaF stimulation, especially the enzyme associated with Golgi membranes. The activities in plasma and microsomal membranes were stimulated by glucagon, whereas the Golgi and nonsedimentable AC were not.
采用埃伦赖希等人的高尔基体分离方法，研究了大鼠肝脏高尔基体和其他细胞组分中腺苷酸环化酶（AC）的分布。在肝脏匀浆中，发现AC活性随时间衰减，但添加1 mM乙二醇双四乙酸（EGTA）可降低酶活性丧失的速率。在高尔基体分离方法最初的两个离心步骤所用的蔗糖培养基中加入1 mM EGTA，可在整个过程中稳定酶活性，并实现良好的酶回收率。在这类制剂中，已证明AC活性不仅与质膜有关，而且还与高尔基体膜和光滑微粒体膜有关。此外，在所采用的条件下，酶活性还与105,000 g×90分钟的上清液组分有关。发现肝脏匀浆的比活性为2.9 pmol·mg蛋白⁻¹·min⁻¹，不可沉淀和微粒体活性处于相同数量级，但高尔基体和质膜活性要高得多。质膜AC的比活性为29 pmol·mg蛋白⁻¹·min⁻¹。高尔基体活性在三个组分中有所不同，当对高尔基体活性进行含量蛋白校正时，GF1中的活性最高（14 pmol），GF2中最低（1.8），GF3中为中等活性（5.5），此时GF1中的活性比质膜高得多（9倍），而GF2和GF3中的活性与质膜相当。在所研究的所有位置，AC对氟化钠刺激敏感，尤其是与高尔基体膜相关的酶。胰高血糖素可刺激质膜和微粒体膜中的活性，而高尔基体和不可沉淀的AC则不受刺激。
