The amino acid sequence of rat liver glucokinase deduced from cloned cDNA.
The amino acid sequence of rat liver glucokinase deduced from cloned cDNA.
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发表时间：
1989-01
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通讯作者：
T. Andreone;R. Printz;S. Pilkis;M. Magnuson;D. Granner
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作者：
T. Andreone;R. Printz;S. Pilkis;M. Magnuson;D. Granner
将大鼠肝葡糖激酶（ATP：D-己糖6-磷酸转移酶，EC 2.7.1.1）纯化至均一，切割，并进行氨基酸序列分析。获得了45%的蛋白质序列，并将这些信息用于设计寡核苷酸探针以筛选大鼠肝脏cDNA文库。1601个碱基对的cDNA（GK 1）含有一个开放的阅读框架，该框架编码在用于产生寡核苷酸探针的肽中发现的氨基酸序列。随后鉴定了第二个cDNA（GK.Z2），其为2346个碱基对长并且对应于几乎整个葡糖激酶mRNA。肝RNA的印迹转移分析表明，葡萄糖激酶mRNA以约2400个核苷酸的单一种类存在。对糖尿病大鼠进行4小时的胰岛素治疗导致该mRNA的诱导增加了30倍。GK.Z2有一个长的开放阅读框架，与已知的部分肽序列，使我们能够推断葡萄糖激酶的一级结构。该酶由465个氨基酸组成，质量为51，924道尔顿。葡萄糖激酶与大鼠脑己糖激酶I和酵母己糖激酶的羧基末端结构域分别具有53%和33%的氨基酸序列同一性。如果也考虑保守氨基酸替换，则葡糖激酶分别在75%和63%的位置处与这两种酶相似。葡萄糖激酶的推定葡萄糖和ATP结合结构域被确定，这些区域似乎是高度保守的己糖激酶家族的酶。
Rat liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified to homogeneity, cleaved, and subjected to amino acid sequence analysis. Forty-five percent of the protein sequence was obtained, and this information was used to design oligonucleotide probes to screen a rat liver cDNA library. A 1601-base pair cDNA (GK1) contained an open reading frame that encoded the amino acid sequences found in the peptides used to generate the oligonucleotide probes. A second cDNA was subsequently identified (GK.Z2), which is 2346 base pairs long and corresponds to nearly the entire glucokinase mRNA. Blot transfer analysis of hepatic RNA showed that glucokinase mRNA exists as a single species of about 2400 nucleotides. Four hours of insulin treatment of diabetic rats resulted in a 30-fold induction of this mRNA. GK.Z2 has a long open reading frame which, with the known partial peptide sequence, allowed us to deduce the primary structure of glucokinase. The enzyme is composed of 465 amino acids and has a mass of 51,924 daltons. Glucokinase has 53 and 33% amino acid sequence identities with the carboxyl-terminal domains of rat brain hexokinase I and yeast hexokinase, respectively. If conservative amino acid replacements are also considered, glucokinase is similar to these two enzymes at 75 and 63% of positions, respectively. The putative glucose- and ATP-binding domains of glucokinase were identified, and these regions appear to be highly conserved in the hexokinase family of enzymes.