Laxminarayan K M, Chan B K, Tetaz T, Bird P I, Mitchell C A
Department of Medicine, Box Hill Hospital, Monash Medical School, Victoria, Australia.
J Biol Chem. 1994 Jun 24;269(25):17305-10.
Agonist stimulation of cells results in phosphatidylinositol turnover and the generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular calcium. The inositol-polyphosphate 5-phosphatase (5-phosphatase) enzymes hydrolyze Ins(1,4,5)P3 in a signal-terminating reaction. We have isolated a 2.7-kilobase (kb) composite cDNA, encoding the 43-kDa membrane-associated 5-phosphatase, by screening a human placental lambda gt11 library, using degenerate oligonucleotides. The 2.7-kb cDNA contains a 1.1-kb open reading frame, comprising 363 amino acids, which encodes a protein of a predicted molecular mass of 42 kDa. Amino acid sequence analysis demonstrates a number of potential sites for phosphorylation by protein kinase C and a CAAX motif in the COOH terminus, which may mediate membrane localization. The recombinant enzyme was expressed in COS-7 cells, resulting in a 50-fold increase in enzyme activity in the detergent-soluble membrane fraction of the cell (nanomole of Ins(1,4,5)P3 hydrolyzed per min/mg), but only a 2.5-fold increase in 5-phosphatase activity in the total cell homogenate. Sequence analysis demonstrated a 73-amino acid domain in the COOH terminus of the 43-kDa membrane-associated 5-phosphatase, which had 30% sequence identity and 67% similarity to a region in the 75-kDa 5-phosphatase and 34% identity and 70% similarity to a sequence in the protein that is encoded by the gene, defective in Lowe's oculocerebrorenal syndrome. As shown by RNA analysis the 43-kDa membrane-associated 5-phosphatase appears to be predominantly expressed in heart, brain, and skeletal muscle.
激动剂刺激细胞会导致磷脂酰肌醇周转并生成肌醇1,4,5 - 三磷酸（Ins(1,4,5)P3），后者可动员细胞内钙。肌醇多磷酸5 - 磷酸酶（5 - 磷酸酶）通过信号终止反应水解Ins(1,4,5)P3。我们通过使用简并寡核苷酸筛选人胎盘λgt11文库，分离出了一个2.7千碱基（kb）的复合cDNA，其编码43 kDa的膜相关5 - 磷酸酶。这个2.7 kb的cDNA包含一个1.1 kb的开放阅读框，由363个氨基酸组成，编码一个预测分子量为42 kDa的蛋白质。氨基酸序列分析显示了多个蛋白激酶C磷酸化的潜在位点以及COOH末端的CAAX基序，这可能介导膜定位。重组酶在COS - 7细胞中表达，导致细胞去污剂可溶膜部分的酶活性增加50倍（每分钟水解的Ins(1,4,5)P3纳摩尔数/毫克），但在总细胞匀浆中5 - 磷酸酶活性仅增加2.5倍。序列分析表明，43 kDa膜相关5 - 磷酸酶的COOH末端有一个73个氨基酸的结构域，与75 kDa 5 - 磷酸酶的一个区域有30%的序列同一性和67%的相似性，与由Lowe眼脑肾综合征缺陷基因编码的蛋白质序列有34%的同一性和70%的相似性。RNA分析表明，43 kDa膜相关5 - 磷酸酶似乎主要在心脏、大脑和骨骼肌中表达。
