Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles.
Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles.
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DOI：
10.1016/s0021-9258(18)42706-8
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发表时间：
1992-03
期刊：
影响因子：
--
通讯作者：
J. Sirois;JoAnne;Richards
中科院分区：
文献类型：
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作者：
J. Sirois;JoAnne;Richards
为了纯化和鉴定人绒毛膜促性腺激素诱导的大鼠排卵前卵泡颗粒细胞中前列腺素内过氧化物合酶（rPGSi）的亚型，将溶解的细胞提取物进行阴离子交换层析，通过一维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离柱组分，并通过免疫印迹和银染技术观察rPGSi。免疫反应性rPGSi和过氧化物酶活性在pH 6.5和6.0下共洗脱。三条免疫反应性rPGSi条带（Mr = 72，000、70，000和59，000）的氨基末端氨基酸序列相同，Mr = 59，000为蛋白水解片段。与绵羊PGS（oPGS）的氨基末端序列的rPGSi的比对表明，只有15的26个残基是相同的（58%的同一性）。与此相反，rPGSi是密切相关的推导的氨基酸序列从鸡和小鼠细胞系中分离的PGS-related cDNA克隆，77%（20/26残基）和96%（25/26残基）的同一性，分别。而oPGS的胰蛋白酶解酶产生Mr = 38，000和Mr = 33，000的片段，仅出现从rPGSi的羧基末端切割的小肽。部分纯化的rPGSi的过氧化物酶活性表现出较低的表观Km和最大速率比oPGS。总的来说，这些结果证明了存在一种新的大鼠PGS同种型（基于纯化、酶活性和氨基末端氨基酸序列），其是卵巢诱导的，并且对于已知的生物学过程排卵是必需的。
To purify and characterize the isoform of prostaglandin endoperoxide synthase (rPGSi) induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles, solubilized cell extracts were subjected to anionic exchange chromatography, column fractions were resolved by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and rPGSi was visualized by immunoblotting and silver staining techniques. Immunoreactive rPGSi and peroxidase activity co-eluted at pH 6.5 and 6.0. Amino-terminal amino acid sequence of three immunoreactive rPGSi bands (Mr = 72,000, 70,000, and 59,000) were identical, Mr = 59,000 being a proteolytic fragment. Alignment of the amino-terminal sequences of rPGSi with those of ovine PGS (oPGS) indicated that only 15 of 26 residues were identical (58% identity). In contrast, rPGSi was closely related to the deduced amino acid sequence of PGS-related cDNA clones isolated from chicken and mouse cell lines, with 77% (20/26 residues) and 96% (25/26 residues) identity, respectively. Whereas tryptic digests of oPGS generated fragments of Mr = 38,000 and Mr = 33,000, only a small peptide appeared cleaved from the carboxyl terminus of rPGSi. Peroxidase activity of partially purified rPGSi exhibited lower apparent Km and maximal velocity than that of oPGS. Collectively, these results document the existence of a novel rat PGS isoform (based on purification, enzymatic activity, and amino-terminal amino acid sequence) which is hormonally induced and obligatory for a known biological process, ovulation.