York J D, Majerus P W
Washington University School of Medicine, Division of Hematology-Oncology, St. Louis, MO 63110.
Proc Natl Acad Sci U S A. 1990 Dec;87(24):9548-52. doi: 10.1073/pnas.87.24.9548.
Inositol polyphosphate 1-phosphatase, an enzyme of the phosphatidylinositol signaling pathway, catalyzes the hydrolysis of the 1-position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. The protein was isolated from calf brain and digested with trypsin or CNBr, and the amino acid sequence of several peptides was determined. Degenerate oligonucleotide primers were designed from amino acid sequence and used to synthesize an 80-base-pair (bp) fragment by the polymerase chain reaction. This product was used to isolate a 1.6-kbp cDNA with an open reading frame of 400 amino acids, 185 bp of 5' untranslated region, and 171 bp of 3' untranslated region followed by a putative poly(A) tail. The coding region of the cDNA was inserted into an expression vector that was used to obtain the recombinant protein from Escherichia coli cells. The recombinant enzyme (44 kDa) had a specific activity and other properties similar to those of native bovine brain inositol polyphosphate 1-phosphatase. It hydrolyzed both inositol phosphate substrates and was inhibited by lithium ions. The enzyme shows minimal sequence similarity to inositol monophosphate phosphatase, the other enzyme inhibited by lithium ions in the signaling pathway.
肌醇多磷酸1-磷酸酶是磷脂酰肌醇信号通路中的一种酶，催化从1,3,4-三磷酸肌醇和1,4-二磷酸肌醇上水解1位磷酸。该蛋白质从小牛脑中分离出来，用胰蛋白酶或溴化氰消化，并测定了几个肽段的氨基酸序列。根据氨基酸序列设计了简并寡核苷酸引物，并通过聚合酶链反应用于合成一个80碱基对（bp）的片段。该产物用于分离一个1.6kbp的cDNA，其开放阅读框为400个氨基酸，5'非翻译区为185bp，3'非翻译区为171bp，后面跟着一个推测的聚腺苷酸尾巴。将cDNA的编码区插入表达载体，用于从大肠杆菌细胞中获得重组蛋白。重组酶（44kDa）具有与天然牛脑肌醇多磷酸1-磷酸酶相似的比活性和其他特性。它能水解两种肌醇磷酸底物，并受到锂离子的抑制。该酶与肌醇单磷酸磷酸酶的序列相似性最小，后者是信号通路中另一种受锂离子抑制的酶。
