Endocrine Cells and its Amidating Capability

大多数多肽激素最初是以前体的形式合成的，需要翻译后加工才能产生生物活性多肽。一般来说，这些步骤包括在成对的碱性残基上进行胰酶切割，随后通过羧基肽酶H去除它们，并通过肽基-甘氨酰-α-酰胺化单加氧酶的作用形成羧基末端酰胺部分。通过这最后一步，它们的羧基末端的酰胺化大多数多肽激素表达了它们的全部生物活性。因此，多肽的α-酰胺化能力是分泌中间多肽的内分泌细胞的关键功能。然而，一些内分泌细胞，如垂体前叶促生长激素细胞，会产生不受抑制的多肽激素。因此，我们进行了这些研究，以探索表达中间酶的可能性是所有内分泌细胞的共同特征，还是仅限于分泌中间激素的细胞的特征。在我们研究中，我们使用了系列…更多的S细胞系，包括来自非内分泌细胞的细胞系(来自NIH3T3成纤维细胞的PSI2细胞和来自小鼠肾脏的BHK细胞)，来自分泌内分泌多肽的细胞(来自大鼠嗜铬细胞瘤的PC-L 2细胞，来自小鼠垂体前叶肿瘤的AtT20细胞和来自大鼠胰岛素瘤的RINm5F细胞)，以及那些未知分泌多肽的内分泌细胞(来自大鼠垂体前叶肿瘤的GH3细胞)。通过测定合成底物D-Tyr-Val-Glyto D-Tyr-Val-NH2的化学转化率，直接测定细胞匀浆中的酰胺化活性。虽然所有的内分泌细胞株都表现出可测量的活性，但非内分泌细胞没有。为了进一步证实这些发现，我们使用逆转录病毒表达载体pZip-Neo-SV(X)转化了不同的细胞系，该载体允许同时表达cdna插入和新霉素抗性基因。为了表达，我们使用了一个人胰腺多肽(PP)CDNA插入。由于PP前驱体含有一个酰胺化中心，我们可以用区域特异性抗血清S11(识别PP的酰胺化羧基末端)和S3(识别PP的中间部分)通过放射免疫分析来测量前驱体产生的酰胺化PP。虽然两个非内分泌细胞株都表达PP前体，但没有检测到羧基末端酰胺化的证据。相反，所有四个内分泌细胞系都产生了酰胺化的PP分子形式，其中主要的一种与合成的PP在Sephadex G-50层析中共洗脱。我们的数据表明，肽α-酰胺化活性并不是所有类型细胞的共同属性，而是内分泌细胞的基本特征，无论它们的天然多肽产品是什么。较少

Most of peptide hormones are initially synthesized as propeptides that require post-translational processing to produce biologically active peptides. Generally these steps include tryptic cleavage at paired basic residues, their subsequent removal by carboxypeptidase H, and formation of a carboxyl terminal amide moiety via the action of peptidyl-glycyl alpha-amidating monooxygenase. By this last step, amidation of their carboxyl terminus most of peptide hormones express their full biological activity. Thus, peptide alpha-amidation capability is a critical function of endocrine cells that secrete amidsted peptides. However, some endocrine cells such as anterior pituitary somatotroph cells produce peptide hormones that are not amidsted. We undertook these studies, therefore, to investigate whither the potential to express the amidsting enzyme is a characteristic common to all endocrine cells 'or a feature restricted to cells that secrete amidsted hormones. For our studies we used a serie … More s of cell lines, including those that originated from non-endocrine cells (psi2 cells derived from NIH3T3 fibroblasts and BHK cells derived ftom baby hamster kidney), from endocrine cells that secrete amidsted peptides (PC-l 2 cells derived from a rat pheochromocytoma, AtT20 cells derived from a mouse anterior pituitary tumor and RINm5F cells derived from a rat insulinoma), and endocrine cells not known to secrete amidsted peptides (GH3 cells derived from a rat anterior pituitary tumor). Amidation activity in cell homogenates was assayed directly by measuring the chemical conversion of a synthetic substrate D-Tyr-Val-Glyto D-Tyr-Val-NH2. Although all of the endocrine cell lines demonstrated measurable activity, the non-endocrine cells did not. For further confirmation of these findings, we transformed the various cell lines using a retroviral expression vector pZip-Neo-SV (X) which permits the simultaneous expression of a cDNA insert and a neomycine resistant gene. For expression, we utilized a human pancreatic polypeptide (PP) CDNA insert. Since the PP precursor contains an amidation site, we could measure the production of amidsted PP from the precursor by radioimmunoassay using region specific antisera S1 1 (which recognizes the amidated carboxyl terminus of PP) and S3 (which recognizes the midportion of PP). Although both non-endocrine cell lines expressed the PP precursor, no evidence of carboxylterminal amidation was detected. In contrast, all four endocrine cell lines produced amidated molecular forms of PP, the principal one co-eluted with synthetic PP on Sephadex G-50 chromatography. Our data suggest that peptide a-amidating activity is not a property common to all cell types but is a fundamental characteristic of endocrine cells, regardless of their natural peptide products. Less