Study on Phosphorylating Sites of Membrane-Bound Guanylyl Cyclase

膜结合鸟苷酸环化酶磷酸化位点的研究

• 批准号: 06454025
• 负责人: SUZUKI Norio
• 金额: $ 4.61万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454025/
• 关键词: Membrane-bound guanylyl cyclase; cGMP; Phosphorylated peptide; Phosphoserine; Spermatozoa; Sea urchin; Signal transduction; S-ethylcysteine; リン酸化部位; バフンウニ; アミノ酸分析

Guanylyl cyclase is found in various cellular compartments as soluble and/or particulate forms and catalyzes the formation of cGMP and inorganic pyrophosphate from GTP.cGMP concentrations in cells have long been known to increase to a wide variety of agents. The transient increases in cGMP concentrations in sea urchin spermatozoa have been explained by transient activation and subsequent inactivation of the guanylyl cyclase, which is closely linked to the state of phosphorylation of the enzyme. Therefore, it is important to identify phosphorylating sites in the guanylyl cyclase. In this project, we developed a large scale purification method of the phosphorylated (131kDa) and dephosphorylated (128kDa) forms of the membrane-bound guanylyl cyclase from spermatozoa of the sea urchin Hemicentrotus pulcherrimus. The purified 131kDa and 128kda forms of the guanylyl cyclase contained 26.0(]SY.+-.])1.3 and 4.3(]SY.+-.])0.7 moles of phosphate per mol protein, respectively. We isolated two phosphorylated peptides from lysyl endopepptidase digest of the 131kDa form of the guanylyl cyclase. One (GSLQDILENDDIK) corresponds to residus from 651 to 663 (kinase-like domain) and other one (TEQLLHRMLPPSIASQLIK) to residues from 883 to 901 (catalytic domain).

鸟苷酸环化酶以可溶性和/或颗粒状形式存在于细胞内，催化GTP合成cGMP和无机焦磷酸。长期以来，cGMP在细胞中的浓度一直被认为是增加到各种介质中的。海胆精子中cGMP浓度的短暂升高可以通过鸟苷酸环化酶的瞬时激活和随后的失活来解释，鸟苷酸环化酶与酶的磷酸化状态密切相关。因此，确定鸟苷酸环化酶中的磷酸化位点是很重要的。在本项目中，我们开发了一种大规模纯化海胆精子膜结合鸟苷酸环化酶的方法，该酶的磷酸化形式(131 KDa)和去磷酸化形式(128 KDa)。纯化的131kDa和128kDa鸟苷酸环化酶每摩尔蛋白分别含有26.0(Sy.+-.)1.3和4.3(Sy.+-.)0.7摩尔磷酸盐。我们从131kDa的鸟苷酸环化酶的赖氨酰内肽酶中分离到两个磷酸化的多肽。另一个(TEQLLHRMLPPSIASQLIK)对应于883~901残基(催化结构域)。

项目成果

Kohji Nomura and Norio Suzuki: "Sea urchin ovoperoxidase : Solubilization and isolation from the fertilization envelope, some structural and functional properties, and degradation by hatching enzyme." Arch. Biochem. Biophys. 319. 525-534 (1995)

Kohji Nomura 和 Norio Suzuki：“海胆卵过氧化物酶：从受精包膜中溶解和分离，一些结构和功能特性，以及孵化酶的降解。”

Abe,H.,Kinoh,H.& Suzuki,N.: "Seasonal variations in the production of the egg-jelly macro molecule,a fucose sulfate glycoconjugate,by the accessory cells" Roux′s Arch.Dev.Biol.,. 203. 402-410 (1994)

Abe, H.、Kinoh, H. 和 Suzuki, N.：“辅助细胞产生蛋冻大分子（一种岩藻糖硫酸糖复合物）的季节变化”Roux 的 Arch.Dev.Biol.,。 203. 402-410 (1994)

Hiroaki Kinoh, Takeshi Shimizu, Hirotaka Fujimoto and Norio Suzuki: "Expression of a putative precursor mRNA for sperm-activating peptide I in accessory cells of the ovary in the sea urchin Hemicentrotus pulcherrimus." Roux's Arch. Dev. Biol.203. 381-388

Hiroaki Kinoh、Takeshi Shimizu、Hirotaka Fujimoto 和 Norio Suzuki：“精子激活肽 I 的推定前体 mRNA 在海胆 Hemicentrotus pulcherrimus 卵巢辅助细胞中的表达。”

Norio Suzuki: "Structure,function and biosynthesis of sperm-activating peptides and fucose sulfate glycoconiugate in the extracellular coat of sea urchin eggs." Zool.Sci.12. 13-27 (1995)

Norio Suzuki：“海胆卵细胞外衣中精子激活肽和岩藻糖硫酸糖复合物的结构、功能和生物合成。”

Shimizu,T.,Yoshino,K.,& Suzuki,N.: "Identification and characterization of putative receptors for sperm-activating peptide I (SAP-I) in spermatozoa" Develop.Growth & Differ.,. 36. 209-221 (1994)

清水 T.、吉野 K.、