Bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase: complete amino acid sequence and localization of phosphorylation sites.
Bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase: complete amino acid sequence and localization of phosphorylation sites.
复制标题
牛心果糖-6-磷酸 2-激酶/果糖-2,6-二磷酸酶：完整的氨基酸序列和磷酸化位点的定位。
DOI：
10.1073/pnas.87.13.4951
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发表时间：
1990
影响因子：
11.1
通讯作者：
Uyeda,K
中科院分区：
文献类型：
--
作者：
Sakata,J;Uyeda,K
我们之前已经证明，牛心果糖-6-磷酸2-激酶/果糖-2,6-二磷酸酶（EC 2.7.1.105/3.1.3.46）被cAMP依赖性蛋白激酶和蛋白激酶C磷酸化；磷酸化导致激酶激活。心脏酶的这种激活与肝脏同工酶的结果相反，在肝脏同工酶中，cAMP依赖性蛋白激酶的磷酸化抑制了激酶活性。作为了解同工酶之间差异的第一步，我们确定了心脏酶的 DNA 序列，并分析了氨基酸序列，特别强调磷酸化位点的位置。我们分离并测序了两个重叠的cDNA片段，它们共同编码了牛心果糖6-磷酸2-激酶/果糖-2,6-二磷酸酶的完整氨基酸序列，这是一种由530个氨基酸组成的蛋白质，计算分子量为60,679。由于推导的蛋白质包含与来自该酶的四种已知胰蛋白酶肽的序列相同的氨基酸序列，因此我们得出结论，推导的蛋白质序列确实代表牛心脏酶。此外，cDNA 片段与来自牛心脏的 4 KB mRNA 杂交。心脏酶的磷酸化位点位于 C 末端附近，而肝脏同工酶的磷酸化位点位于 N 末端附近。磷酸化位点的这些相反位置可以解释共价修饰对酶活性的对比作用。
We have shown previously that bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) is phosphorylated by cAMP-dependent protein kinase and protein kinase C; phosphorylation results in activation of kinase. This activation of heart enzyme is in contrast to results with the liver isozyme, in which phosphorylation by cAMP-dependent protein kinase inhibits the kinase activity. As an initial step toward understanding this difference between the isozymes we have determined the DNA sequence of the heart enzyme and analyzed the amino acid sequence with special emphasis on the location of the phosphorylation site. We isolated and sequenced two overlapping cDNA fragments, which together could encode the complete amino acid sequence of bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase, a protein of 530 amino acids, with a calculated molecular weight of 60,679. Since the deduced protein contained amino acid sequences identical to the sequences of four known tryptic peptides from this enzyme we concluded that the deduced protein sequence did represent bovine heart enzyme. In addition, a cDNA fragment hybridized to a 4-kilobase mRNA from bovine heart. The phosphorylation sites of the heart enzyme were located near the C terminus, whereas the phosphorylation site of the liver isozyme is known to be located near the N terminus. These opposite locations of the phosphorylation sites may explain the contrasting effect of the covalent modification on the enzymes' activities.