Fricker L D, Devi L
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461.
J Neurochem. 1993 Oct;61(4):1404-15. doi: 10.1111/j.1471-4159.1993.tb13634.x.
Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, "proCPE." To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [35S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37 degrees C but was unaffected when cells were chased at either 20 or 15 degrees C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCl2. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.
羧肽酶E（CPE）在肽类激素和神经递质的翻译后加工过程中发挥作用。与其他肽加工酶一样，CPE以可溶性和膜相关形式存在于分泌颗粒中，这两种形式均源自单一前体“前CPE”的翻译后加工。为了确定前CPE加工的细胞内位点，我们在源自小鼠垂体前叶的细胞系AtT-20中研究了其生物合成和翻译后加工过程。用[35S]甲硫氨酸脉冲处理15分钟后，鉴定出了可溶性和膜结合形式的CPE，这表明产生这些CPE形式的翻译后加工事件发生在内质网或早期高尔基体中。当细胞在37℃下追踪2小时时，可溶性和膜结合形式的CPE的相对比例发生了变化，但在20℃或15℃下追踪时则不受影响，这表明膜形式向可溶性形式的进一步加工发生在高尔基体后区室中。用氯喹处理细胞并没有改变可溶性和膜形式的相对分布，这表明该加工事件不需要酸性区室。CPE的过表达并没有影响可溶性和膜结合形式的CPE的分布，这表明CPE加工酶不是限速酶。为了直接检测CPE加工酶，我们分离了牛垂体前叶分泌囊泡。在纯化的分泌囊泡膜中检测到了一种释放膜结合形式CPE的酶活性。这种去除CPE C末端区域的酶受到EDTA和苯甲基磺酰氟的部分抑制，并被CaCl2激活。总之，这些数据表明CPE的翻译后加工发生在分泌颗粒中，并且这种活性可能由一种激素原转化酶样酶介导。
