Bausch-Jurken M T, Mahnke-Zizelman D K, Morisaki T, Sabina R L
Department of Cellular Biology and Anatomy, Medical College of Wisconsin, Milwaukee 53226.
J Biol Chem. 1992 Nov 5;267(31):22407-13.
Human AMPD2 cDNA clones have been isolated from T-lymphoblast and placental lambda gt11 libraries utilizing a previously cloned rat partial AMPD2 cDNA as the probe. Alignment analysis of all cDNA clones indicates the presence of intervening sequences in several placental isolates. This has been confirmed by sequencing human AMPD2 genomic clones. Intervening sequences can be removed from the cDNA clones by restriction with endonucleases at unique sites within the proposed open reading frame. This results in a 3292-base pair cDNA proposed to contain the entire AMPD2 open reading frame, which would encode a 760-amino acid polypeptide with a predicted subunit molecular mass of 88.1 kDa. Nucleotide and predicted amino acid comparisons with the 264 base pairs of proposed coding sequences in the rat AMPD2 cDNA demonstrate 91% similarity and identity, respectively. A comparison of the predicted human AMPD1 and AMPD2 polypeptides demonstrates homology in their C-terminal domains. Included in this region is the conserved motif, SLSTDDP, proposed to be part of the catalytic site of all AMP deaminases. In contrast, the predicted N-terminal domains of the human AMPD1 and AMPD2 polypeptides are unique. When placed in a prokaryotic expression vector, the human AMPD2 cDNA expresses AMP deaminase activity which can be precipitated with polyclonal antisera specific for isoform L.
利用先前克隆的大鼠AMPD2部分cDNA作为探针，已从T淋巴细胞和成胎盘λgt11文库中分离出人AMPD2 cDNA克隆。对所有cDNA克隆的比对分析表明，几个胎盘分离株中存在间隔序列。对人AMPD2基因组克隆进行测序已证实了这一点。通过在所提议的开放阅读框内的独特位点用核酸内切酶进行酶切，可以从cDNA克隆中去除间隔序列。这产生了一个3292碱基对的cDNA，该cDNA被认为包含整个AMPD2开放阅读框，它将编码一个760个氨基酸的多肽，预测亚基分子量为88.1 kDa。与大鼠AMPD2 cDNA中264个碱基对的提议编码序列进行核苷酸和预测氨基酸比较，分别显示出91%的相似性和同一性。对预测的人AMPD1和AMPD2多肽进行比较，发现它们的C末端结构域具有同源性。该区域包括保守基序SLSTDDP，该基序被认为是所有AMP脱氨酶催化位点的一部分。相比之下，人AMPD1和AMPD2多肽的预测N末端结构域是独特的。当置于原核表达载体中时，人AMPD2 cDNA表达的AMP脱氨酶活性可被针对同工型L的多克隆抗血清沉淀。
