Differential processing of proenkephalin by prohormone convertases 1(3) and 2 and furin.
Differential processing of proenkephalin by prohormone convertases 1(3) and 2 and furin.
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DOI：
10.1016/s0021-9258(19)74221-5
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发表时间：
1993-12
期刊：
影响因子：
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通讯作者：
M. Breslin;I. Lindberg;S. Benjannet;J P Mathis;C. Lazure;N. G. Seidah
中科院分区：
文献类型：
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作者：
M. Breslin;I. Lindberg;S. Benjannet;J P Mathis;C. Lazure;N. G. Seidah
重组牛痘病毒载体被用来共表达小鼠激素原转化酶1（mPC 1），mPC 2，或人类弗林蛋白酶与人类前脑啡肽原在GH4 C1细胞（大鼠垂体somatomammotrophs）检查这些酶的蛋白水解加工的前脑啡肽原。从GH4 C1细胞和相应的条件培养基中获得的高压凝胶渗透大小分级提取物进行放射免疫测定，揭示了由每种酶产生的产物的免疫反应性的不同配置文件。PC 1产生中等大小的加工产物（3-10 kDa）;在肽B、5.3-kDa片段和游离Leu 5-脑啡肽的位置处观察到洗脱的主要免疫反应性含脑啡肽物质。PC 2表现出更完整的加工曲线。产生的主要免疫反应性脑啡肽是游离Met 5-脑啡肽-Arg-Phe、游离Met 5-脑啡肽-Arg-Gly-Leu、游离Leu 5-脑啡肽和游离Met 5-脑啡肽。因此，PC 2似乎比PC 1更能从前脑啡肽产生活性阿片单位。最后，弗林蛋白酶裂解脑啡肽原，生成肽B，一个未鉴定的峰，位于18-和5.3-kDa片段之间，以及少量的5.3-kDa片段。放射性测序数据证实，PC 1产生的5.3 kDa片段是Lys-Lys裂解的结果。PC 1在Lys-Lys位点切割前脑啡肽原（但不切割阿黑皮素原）的能力意味着配对碱性切割位点的结构背景在确定切割特异性方面可能比该位点的特定碱性残基对更重要。
Recombinant vaccinia virus vectors were used to coexpress mouse prohormone convertase 1 (mPC1), mPC2, or human furin together with human proenkephalin in GH4C1 cells (rat pituitary somatomammotrophs) to examine the proteolytic processing of proenkephalin by these enzymes. Radioimmunoassays performed on high pressure gel permeation size-fractionated extracts obtained from GH4C1 cells and corresponding conditioned media revealed distinct profiles of immunoreactivity for products generated by each enzyme. PC1 produced intermediate sized processing products (3-10 kDa); the major immunoreactive enkephalin-containing species observed eluted at the positions of peptide B, the 5.3-kDa fragment, and free Leu5-enkephalin. PC2 exhibited a more complete processing profile. The major immunoreactive enkephalins produced were free Met5-enkephalin-Arg-Phe, free Met5-enkephalin-Arg-Gly-Leu, free Leu5-enkephalin, and free Met5-enkephalin. Thus PC2 appears to be more capable of generating active opioid units from proenkephalin than is PC1. Finally, furin cleaved proenkephalin to generate peptide B, an unidentified peak between the 18- and 5.3-kDa fragments, and a small amount of the 5.3-kDa fragment. Radiosequencing data verified that the production of the 5.3-kDa fragment by PC1 occurred as a result of a Lys-Lys cleavage. The ability of PC1 to cleave proenkephalin (but not proopiomelanocortin) at a Lys-Lys site implies that the structural context of the paired basic cleavage site may be more important in the determination of cleavage specificity than the particular pair of basic residues at the site.