Zolkiewska A, Nightingale M S, Moss J
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11352-6. doi: 10.1073/pnas.89.23.11352.
Mono-ADP-ribosylation is a reversible modification of proteins, with NAD:arginine ADP-ribosyltransferases (EC 2.4.2.31) and ADP-ribosylarginine hydrolases (EC 3.2.2.19) catalyzing the opposing reactions in an ADP-ribosylation cycle. A membrane-associated arginine-specific (mono)-ADP-ribosyltransferase was purified 215,000-fold from rabbit skeletal muscle. On the basis of the amino acid sequences of HPLC-purified tryptic peptides, degenerate oligonucleotide primers were synthesized and used in a polymerase chain reaction (PCR)-based procedure to generate cDNA. A specific probe, based on PCR-generated sequence, was used to screen a rabbit skeletal muscle cDNA library. A composite cDNA sequence, obtained from library screening and rapid amplification of the 5' end of the cDNA, contained a 981-base-pair open reading frame, encoding a 36,134-Da protein. The deduced amino acid sequence contained the sequences of the tryptic peptides, hydrophobic amino and carboxyl termini, and two potential sites for N-linked glycosylation. Escherichia coli cells transformed with an expression vector containing transferase-specific sequence expressed ADP-ribosyltransferase activity. A transferase-specific oligonucleotide probe recognized a 4-kilobase mRNA expressed primarily in rabbit skeletal and cardiac muscle. There was no extended similarity in deduced amino acid sequences of the muscle transferase and several bacterial ADP-ribosylating toxins. The hydrophobic amino and carboxyl termini may represent a signal peptide and a site for a glycosyl-phosphatidylinositol anchor, respectively.
单磷酸腺苷核糖基化是一种蛋白质的可逆修饰，烟酰胺腺嘌呤二核苷酸：精氨酸 ADP 核糖基转移酶（EC 2.4.2.31）和 ADP 核糖基精氨酸水解酶（EC 3.2.2.19）在 ADP 核糖基化循环中催化相反的反应。从兔骨骼肌中纯化出一种与膜相关的精氨酸特异性（单）ADP 核糖基转移酶，纯化倍数达 215,000 倍。根据高效液相色谱纯化的胰蛋白酶肽段的氨基酸序列，合成了简并寡核苷酸引物，并用于基于聚合酶链反应（PCR）的方法来生成 cDNA。基于 PCR 生成的序列的特异性探针用于筛选兔骨骼肌 cDNA 文库。从文库筛选和 cDNA 5'端的快速扩增获得的复合 cDNA 序列包含一个 981 个碱基对的开放阅读框，编码一个 36,134 道尔顿的蛋白质。推导的氨基酸序列包含胰蛋白酶肽段的序列、疏水的氨基和羧基末端，以及两个潜在的 N 连接糖基化位点。用含有转移酶特异性序列的表达载体转化的大肠杆菌细胞表达 ADP 核糖基转移酶活性。一种转移酶特异性寡核苷酸探针识别一种主要在兔骨骼肌和心肌中表达的 4 千碱基 mRNA。肌肉转移酶的推导氨基酸序列与几种细菌 ADP 核糖基化毒素没有广泛的相似性。疏水的氨基和羧基末端可能分别代表信号肽和糖基磷脂酰肌醇锚定的位点。
