Nigam S K, Blobel G
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021.
J Biol Chem. 1989 Oct 5;264(28):16927-32.
A canine pancreas homogenate was subfractionated by several differential centrifugation steps. The distribution of cAMP-dependent protein kinase in the various fractions was monitored by assaying [3H]cAMP binding and photo-cross-linking of the regulatory subunits of the enzyme (RI and RII) with radiolabeled 8-azido-cAMP. The distribution of the kinase was also compared to that of markers for the plasma membrane, the endoplasmic reticulum and the cytosol. While our results confirm previous studies suggesting the presence of cyclic AMP-dependent protein kinase in the cytosol and Golgi, a significant amount of the total [3H] cAMP binding and photolabeled R subunits (both RI and RII) were found in rough microsomes (RM). The association is relatively resistant to extraction with EDTA, low and high ionic strength solutions. These extractions unmasked several new phosphorylation substrates in the "stripped" RM that were inaccessible in the RM, possibly because they were covered by ribosomes or peripheral membrane proteins. RII with a molecular mass of 52 kDa (RII-52 kDa) was the predominant RII found in the cytosolic fraction, whereas RII-52 kDa and RII with a molecular mass of 54 kDa (RII-54 kDa) were approximately equally enriched in the RM fraction. The mobility of the RII-52 kDa-photolabeled band could be shifted to the mobility of the RII-54 kDa band by phosphorylation with purified catalytic subunit and ATP, indicating that they represent "dephospho" and "phospho" forms of RII, respectively. A more precise localization to the rough endoplasmic reticulum was accomplished by isopycnic floatation in sucrose gradients. The enzyme cobanded at the density of rough microsomes and shifted to the lower density of "stripped" microsomes after treatment with puromycin/high salt, which specifically removes ribosomes.
通过几个差速离心步骤对犬胰腺匀浆进行亚分级分离。通过检测[3H]cAMP结合以及该酶调节亚基（RI和RII）与放射性标记的8-叠氮基-cAMP的光交联，监测各分级中cAMP依赖性蛋白激酶的分布。还将该激酶的分布与质膜、内质网和胞质溶胶的标志物分布进行了比较。虽然我们的结果证实了先前的研究，表明胞质溶胶和高尔基体中存在环AMP依赖性蛋白激酶，但在粗面微粒体（RM）中发现了大量总的[3H]cAMP结合以及光标记的R亚基（RI和RII）。这种结合相对抗EDTA、低离子强度和高离子强度溶液的提取。这些提取揭示了“剥离”的RM中一些新的磷酸化底物，这些底物在RM中无法接近，可能是因为它们被核糖体或外周膜蛋白覆盖。分子量为52 kDa的RII（RII-52 kDa）是胞质分级中发现的主要RII，而RII-52 kDa和分子量为54 kDa的RII（RII-54 kDa）在RM分级中的富集程度大致相同。用纯化的催化亚基和ATP磷酸化后，RII-52 kDa光标记带的迁移率可转变为RII-54 kDa带的迁移率，表明它们分别代表RII的“去磷酸化”和“磷酸化”形式。通过在蔗糖梯度中进行等密度漂浮实现了更精确地定位于粗面内质网。该酶在粗面微粒体的密度处共带，在用嘌呤霉素/高盐处理后转移到“剥离”微粒体的较低密度处，嘌呤霉素/高盐可特异性去除核糖体。
