Zollinger L, Noël G, Des Parois L, Sales V, Crine P, Boileau G
Département de Biochimie, Faculté de Médecine, Université de Montréal, Qc, Canada.
Mol Cell Endocrinol. 1988 Jul;58(1):31-41. doi: 10.1016/0303-7207(88)90051-2.
Pro-opiomelanocortin (POMC) is the common precursor of several pituitary hormones including alpha-melanotropic hormone, adrenocorticotropic hormone, beta-lipotropin and beta-endorphin. The porcine POMC cDNA was inserted downstream from the late promoter of an SV40-derived expression vector and co-transfected in NIH 3T3 cells with a marker plasmid carrying the neomycin resistance gene. Colonies resistant to the neomycin analog G418 were selected and analyzed for the production of POMC-related peptides by radioimmunoassay. Three clones were found to produce from 350 to 1750 pg of POMC-related peptides per 10(6) cells in 16 h and selected for further analysis. The number of POMC cDNA copies integrated in the host cell genome was determined and the levels of transcription were compared. POMC-related material released in the culture medium by the best producing clone (NJP 4-4) was further analyzed by gel filtration and reversed-phase high-pressure liquid chromatography combined with radioimmunoassays. POMC was found to be synthesized and secreted without further processing or degradation. Negligible amounts of POMC-immunoreactive species were found in cellular extracts indicating that the prohormone is secreted from the NIH 3T3 cells without storage, presumably through a constitutive pathway. Our results suggest that NIH 3T3 fibroblasts do not contain the enzymatic machinery to process complex precursors such as POMC.
阿片促黑激素皮质素原（POMC）是几种垂体激素的共同前体，包括α-促黑素细胞激素、促肾上腺皮质激素、β-促脂素和β-内啡肽。将猪POMC cDNA插入到一个源自SV40的表达载体的晚期启动子下游，并与携带新霉素抗性基因的标记质粒一起共转染到NIH 3T3细胞中。选择对新霉素类似物G418有抗性的菌落，并通过放射免疫测定法分析其POMC相关肽的产生情况。发现有三个克隆在16小时内每10^6个细胞产生350至1750皮克的POMC相关肽，并选择它们进行进一步分析。确定整合到宿主细胞基因组中的POMC cDNA拷贝数，并比较转录水平。通过凝胶过滤和反相高压液相色谱结合放射免疫测定法，对产生量最高的克隆（NJP 4-4）在培养基中释放的POMC相关物质进行了进一步分析。发现POMC是在没有进一步加工或降解的情况下合成并分泌的。在细胞提取物中发现的POMC免疫反应性物质的量可以忽略不计，这表明前体激素是从NIH 3T3细胞中分泌出来的，没有储存，大概是通过组成型途径。我们的结果表明，NIH 3T3成纤维细胞不含有处理诸如POMC等复杂前体的酶机制。
