MOLECULAR-CLONING AND SEQUENCE-ANALYSIS OF THE CATALYTIC SUBUNIT OF BOVINE TYPE-2A PROTEIN PHOSPHATASE
MOLECULAR-CLONING AND SEQUENCE-ANALYSIS OF THE CATALYTIC SUBUNIT OF BOVINE TYPE-2A PROTEIN PHOSPHATASE
复制标题
DOI：
10.1073/pnas.84.14.4880
复制
发表时间：
1987-07-01
影响因子：
11.1
通讯作者：
MUMBY, MC
中科院分区：
文献类型：
--
作者：
GREEN, DD;YANG, SI;MUMBY, MC
我们已经分离出一个cDNA克隆，对应于牛2A型蛋白磷酸酶的Mr 38，000催化亚基。该cDNA是通过使用寡核苷酸探针从牛肾上腺cDNA文库中分离出来的，该探针的序列基于从纯化的心脏酶的溴化氰片段中获得的部分氨基酸序列。整个1724碱基对的cDNA已被测序，发现含有一个开放阅读框，编码一种325个氨基酸的蛋白质，计算分子量为37，320。推导的氨基酸序列包含实验确定的五种不同溴化氰肽的序列。用克隆的cDNA转染COS-m6细胞导致蛋白质的瞬时表达，所述蛋白质可以通过免疫印迹分析用针对纯化的心脏蛋白磷酸酶的单克隆抗体检测。表达的蛋白质具有相同的表观分子量的纯化酶时，通过NaDodSO 4/聚丙烯酰胺凝胶电泳分析，这表明该克隆包含磷酸酶mRNA的整个编码区。克隆的cDNA与牛心脏和肾上腺中2.0磷酸酶的mRNA杂交。在降低严格性的条件下，cDNA还与心脏组织中的1.2种酶的mRNA种类杂交。
We have isolated a cDNA clone corresponding to the Mr 38,000 catalytic subunit of bovine type 2A protein phosphatase. The cDNA was isolated from a bovine adrenal gland cDNA library through the use of oligonucleotide probes whose sequences were based on partial amino acid sequence obtained from cyanogen bromide fragments of the purified cardiac enzyme. The entire 1724-base-pair cDNA has been sequenced and found to contain an open reading frame coding for a protein of 325 amino acids having a calculated molecular weight of 37,320. The deduced amino acid sequence contains the experimentally determined sequences of five different cyanogen bromide peptides. Transfection of COS-m6 cells with the cloned cDNA resulted in transient expression of a protein that could be detected by immunoblot analysis with a monoclonal antibody directed against the purified cardiac protein phosphatase. The expressed protein had the same apparent molecular weight as the purified enzyme when analyzed by NaDodSO4/polyacrylamide gel electrophoresis, suggesting that this clone contains the entire coding region of the phosphatase mRNA. The cloned cDNA hybridizes to a mRNA of 2.0 kilobases in bovine heart and adrenal gland. Under conditions of reduced stringency, the cDNA also hybridizes to a mRNA species of 1.2 kilobases in cardiac tissue.