Stoffers D A, Ouafik L, Eipper B A
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1991 Jan 25;266(3):1701-7.
The COOH-terminal alpha-amidation of bioactive peptides is a 2-step process catalyzed by two separable enzymatic activities both derived from the peptidylglycine alpha-amidating monooxygenase (PAM) precursor. Two forms of PAM mRNA (rPAM-1 and -2), differing by the presence or absence of optional Exon A, were previously characterized; both encode precursors predicted to have an NH2-terminal signal sequence, an intragranular domain containing both enzymatic activities, and a single transmembrane domain followed by a short, cytoplasmic COOH-terminal domain. In this report, two novel types of PAM mRNA were identified in adult rat atrium. A cDNA of each type was sequenced, and the results indicate that rPAM-3 and -4 could be related to each other and to the previously characterized rat PAM cDNAs by alternative mRNA splicing. Deletion of a 258-nucleotide segment (optional Exon B) encoding the transmembrane domain from rPAM-3 and the presence of a novel 3'-exon in rPAM-4 mean that both rPAM-3 and -4 mRNAs encode precursor proteins that have an NH2-terminal signal peptide but lack a transmembrane domain. The rPAM-4 precursor protein lacks the region of the PAM precursor catalyzing the second step in the alpha-amidation reaction. Low levels of rPAM-3 and -4 type mRNA were detected in atrium. Utilizing the polymerase chain reaction, two major patterns of distribution of forms of PAM mRNA were found. In the heart and central nervous system, PAM mRNAs both containing and lacking optional Exon A were prevalent and almost all of the PAM mRNAs detected contained optional Exon B. In the pituitary and submaxillary glands, PAM mRNAs lacking optimal Exon A were prevalent, as were PAM mRNAs lacking all or part of optional Exon B. Since the distribution of PAM activity between soluble and membrane fractions is tissue-specific and developmentally regulated and since rPAM-4 lacks an enzymatic portion of the PAM precursor, the tissue-specific expression of these forms of rat PAM mRNA is expected to be of functional significance.
生物活性肽的羧基末端α-酰胺化是一个由两种可分离的酶活性催化的两步过程，这两种酶活性均来源于肽基甘氨酸α-酰胺化单加氧酶（PAM）前体。先前已鉴定出两种形式的PAM mRNA（rPAM - 1和 - 2），它们因是否存在选择性外显子A而有所不同；两者都编码预测具有NH2末端信号序列、包含两种酶活性的颗粒内结构域、单个跨膜结构域以及紧随其后的短细胞质羧基末端结构域的前体。在本报告中，在成年大鼠心房中鉴定出两种新型的PAM mRNA。对每种类型的cDNA进行了测序，结果表明rPAM - 3和 - 4可能通过可变mRNA剪接相互关联，并与先前鉴定的大鼠PAM cDNA相关。从rPAM - 3中缺失编码跨膜结构域的258个核苷酸片段（选择性外显子B）以及rPAM - 4中存在一个新的3'-外显子意味着rPAM - 3和 - 4 mRNA都编码具有NH2末端信号肽但缺乏跨膜结构域的前体蛋白。rPAM - 4前体蛋白缺乏PAM前体催化α-酰胺化反应第二步的区域。在心房中检测到低水平的rPAM - 3和 - 4型mRNA。利用聚合酶链反应，发现了PAM mRNA形式的两种主要分布模式。在心脏和中枢神经系统中，包含和缺乏选择性外显子A的PAM mRNA都很普遍，并且几乎所有检测到的PAM mRNA都包含选择性外显子B。在垂体和颌下腺中，缺乏最佳外显子A的PAM mRNA很普遍，缺乏全部或部分选择性外显子B的PAM mRNA也是如此。由于PAM活性在可溶性和膜部分之间的分布具有组织特异性且受发育调控，并且由于rPAM - 4缺乏PAM前体的酶促部分，预计这些形式的大鼠PAM mRNA的组织特异性表达具有功能意义。
