Chang T L, Loh Y P
Endocrinology. 1984 Jun;114(6):2092-9. doi: 10.1210/endo-114-6-2092.
Secretory granules (SGs) from rat intermediate lobes (IL) were isolated in a highly purified form by differential centrifugation, followed by sucrose density gradient centrifugation. The purified IL-SGs were lysed by freezing and thawing. The granule lysate was then centrifuged to generate membrane and soluble fractions. Proopiocortin -converting enzyme (PCE) activity was assayed by incubation of [3H]arginine- or [3H] phenylalanine-labeled toad proopiocortin with the total granule lysate, the membrane, or the soluble fraction at pH 5.0. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea-gel electrophoresis. The PCE activity in rat IL-SG lysate cleaved proopiocortin to 21,000 mol wt ACTH, 21,000 mol wt ACTH/lipotropin (LPH), 13,000 mol wt ACTH, beta LPH, beta-endorphin-like peptides, and alpha MSH-like peptides, similar to those synthesized by the toad intermediate lobe in situ. Treatment of the PCE cleavage products with carboxypeptidase B resulted in the liberation of free arginine. This observation together with the nature of the products formed suggest that the PCE activity cleaved at pairs of basic residues of proopiocortin , yielding one or more products that terminated with an arginine or an arginine-lysine. PCE activity was found in membrane and soluble granule fractions, and both activities were inhibited by leupeptin, p-chloromercuribenzoate, dithiodipyridine, and pepstatin A, but not by chloroquine or N-alpha-p-tosyl-L-lysine-chloromethylketone HCl. Diisopropyl fluorophosphate and other thiol protease reagents (p-chloromercuriphenyl sulfonic acid, iodoacetic acid, and HgCl2) had a small inhibitory effect. The products formed by PCE activities in the membrane and soluble fractions were similar to those cleaved by the total granule lysate. The membrane fraction primarily cleaved proopiocortin between ACTH and beta LPH to form 21,000 (21 K) mol wt ACTH and beta-LPH, similar to the first processing step in the IL in situ. The soluble fraction, however, showed a greater tendency to cleave proopiocortin between the 16 K N-terminal glycopeptide and ACTH, to yield twice as much 21 K ACTH/LPH product as the membrane fraction. The membrane-associated PCE activity was found to be easily solubilized by extraction with high salt (1 M NaCl), suggesting that it is not an integral granule membrane protein.
通过差速离心，随后进行蔗糖密度梯度离心，以高度纯化的形式分离出大鼠中间叶（IL）的分泌颗粒（SGs）。纯化后的IL-SGs经冻融裂解。然后将颗粒裂解物离心，以获得膜组分和可溶性组分。通过在pH 5.0条件下，将[3H]精氨酸或[3H]苯丙氨酸标记的蟾蜍阿片促黑激素原与总颗粒裂解物、膜组分或可溶性组分一起温育，来测定阿片促黑激素原转化酶（PCE）的活性。通过用促肾上腺皮质激素（ACTH）和β-内啡肽抗血清进行免疫沉淀，随后进行酸性尿素凝胶电泳，来鉴定加工产物。大鼠IL-SG裂解物中的PCE活性将阿片促黑激素原裂解为21,000分子量的ACTH、21,000分子量的ACTH/促脂素（LPH）、13,000分子量的ACTH、β-LPH、β-内啡肽样肽和α-促黑素细胞激素（α-MSH）样肽，类似于蟾蜍中间叶原位合成的产物。用羧肽酶B处理PCE裂解产物，导致游离精氨酸的释放。这一观察结果连同所形成产物的性质表明，PCE活性在阿片促黑激素原的碱性残基对处裂解，产生一种或多种以精氨酸或精氨酸-赖氨酸结尾的产物。在膜组分和可溶性颗粒组分中均发现了PCE活性，且两种活性均受到亮抑酶肽、对氯汞苯甲酸、二硫代二吡啶和胃蛋白酶抑制剂A的抑制，但不受氯喹或N-α-对甲苯磺酰-L-赖氨酸氯甲基酮盐酸盐的抑制。二异丙基氟磷酸和其他巯基蛋白酶试剂（对氯汞苯磺酸、碘乙酸和HgCl2）具有较小的抑制作用。膜组分和可溶性组分中PCE活性形成的产物与总颗粒裂解物裂解产生的产物相似。膜组分主要在ACTH和β-LPH之间裂解阿片促黑激素原，形成21,000（21K）分子量的ACTH和β-LPH，类似于IL原位的第一步加工步骤。然而，可溶性组分在16K N端糖肽和ACTH之间裂解阿片促黑激素原的倾向更大，产生的21K ACTH/LPH产物是膜组分的两倍。发现与膜相关的PCE活性很容易通过用高盐（1M NaCl）提取而溶解，这表明它不是一种完整的颗粒膜蛋白。
