Molecular cloning and nucleotide sequence of cDNAs encoding the alpha-subunit of human electron transfer flavoprotein.
Molecular cloning and nucleotide sequence of cDNAs encoding the alpha-subunit of human electron transfer flavoprotein.
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DOI：
10.1016/s0021-9258(19)37655-0
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发表时间：
1988-10
期刊：
影响因子：
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通讯作者：
Gaetano FinocchiaroS;Michinori Itog;Yasuyuki Ikedali;Kay Tanaka
中科院分区：
文献类型：
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作者：
Gaetano FinocchiaroS;Michinori Itog;Yasuyuki Ikedali;Kay Tanaka
克隆并测序了人电子转移黄素蛋白(pα-ETF)α亚基前体的cDNA。CDNA跨越1300个碱基对，包括333个氨基酸的整个编码区。通过杂交选择翻译、转录/翻译和线粒体处理这些翻译产生的蛋白质，证实了pα-ETF克隆的同一性。将克隆的氨基酸序列与纯化的大鼠α-亚基电子转移黄素蛋白(α-ETF)制备的7个胰蛋白肽的氨基酸序列进行比对，进一步证实了克隆的同源性。从这些大鼠α-ETF胰蛋白肽中提取的105个氨基酸中，有98个氨基酸与人类cDNA中的氨基酸相匹配。这7个氨基酸的替换可能是由于物种的差异。计算出人α-ETF前体的相对分子质量为35,084。通过氨基末端测序无法确定成熟蛋白的氨基端氨基酸，可能是由于堵塞，因此无法计算成熟α-ETF的准确分子量。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定，前体和成熟α-ETF的分子大小分别为35 kDa和32 kDa。
cDNAs encoding the precursor of the alpha-subunit of human electron transfer flavoprotein (p alpha-ETF) were cloned and sequenced. The cDNAs span 1,300 base pairs and include the entire coding region of 333 amino acids. The identity of the p alpha-ETF clones was confirmed by hybrid selected translation, by transcription/translation of a cDNA, and by mitochondrial processing of the protein produced by these translations. The identity of the cDNA clones was further confirmed by matching the amino acid sequence deduced from the nucleotide sequence of the cDNAs to amino acid sequences determined from seven tryptic peptides prepared from purified rat alpha-subunit of electron transfer flavoprotein (alpha-ETF). Ninety-eight of 105 amino acids from these rat alpha-ETF tryptic peptides matched with those deduced from the human cDNAs. The seven amino acid substitutions are presumably due to species difference. The calculated molecular weight of the human alpha-ETF precursor was 35,084. The amino-terminal amino acid of the mature protein could not be determined by amino-terminal sequencing, presumably due to blockage and, therefore, an accurate molecular weight of mature alpha-ETF could not be calculated. The molecular sizes of the precursor and mature alpha-ETF have been estimated to be 35 and 32 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.