Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis.
Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis.
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DOI：
10.1210/mend-1-11-777
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发表时间：
1987-11
影响因子：
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通讯作者：
B. Eipper;Larry P. Park;Ian M. Dickerson;H. Keutmann;Elizabeth A. Thiele;Henry Rodriguez;P. R. Schofield;R. Mains
中科院分区：
文献类型：
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作者：
B. Eipper;Larry P. Park;Ian M. Dickerson;H. Keutmann;Elizabeth A. Thiele;Henry Rodriguez;P. R. Schofield;R. Mains
许多生物活性肽在其COOH末端以氨基酸α-酰胺终止。负责这种必需的翻译后修饰的酶被称为肽基甘氨酸α-酰胺化单加氧酶或PAM。我们通过使用抗体筛选牛中间垂体λ gt 11表达文库来鉴定编码该酶的cDNA。β-半乳糖苷酶/PAM融合蛋白的抗体从牛垂体匀浆中去除PAM活性。完整cDNA预测的108，207道尔顿蛋白质大小约为纯化PAM的两倍。纯化的PAM的NH 2-末端之前的NH 2-末端信号序列和短的前肽。几个PAM溴化氰肽的序列被定位在预测的蛋白质的NH 2-末端的一半。该cDNA编码一个额外的430个氨基酸的颗粒内结构域，随后是一个假定的跨膜结构域和一个亲水性胞质结构域。从牛垂体神经中间体纯化的PAM形式可以通过在前体中的10对碱性氨基酸的子集处的内切蛋白水解裂解来产生。在牛垂体和大脑皮质中发现高水平的PAM mRNA。在促肾上腺皮质激素肿瘤细胞中，PAM mRNA和促肾上腺皮质激素原/内啡肽mRNA的水平受到糖皮质激素和CRF的平行调节。
Many bioactive peptides terminate with an amino acid alpha-amide at their COOH terminus. The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine alpha-amidating monooxygenase or PAM. We identified cDNAs encoding the enzyme by using antibodies to screen a bovine intermediate pituitary lambda gt11 expression library. Antibodies to a beta-galactosidase/PAM fusion protein removed PAM activity from bovine pituitary homogenates. The 108,207 dalton protein predicted by the complete cDNA is approximately twice the size of purified PAM. An NH2-terminal signal sequence and short propeptide precede the NH2 terminus of purified PAM. The sequences of several PAM cyanogen bromide peptides were localized in the NH2-terminal half of the predicted protein. The cDNA encodes an additional 430 amino acid intragranular domain followed by a putative membrane spanning domain and a hydrophilic cytoplasmic domain. The forms of PAM purified from bovine neurointermediate pituitary may be generated by endoproteolytic cleavage at a subset of the 10 pairs of basic amino acids in the precursor. High levels of PAM mRNA were found in bovine pituitary and cerebral cortex. In corticotropic tumor cells, levels of PAM mRNA and pro-ACTH/endorphin mRNA were regulated in parallel by glucocorticoids and CRF.