Study on Phosphorylating Sites of Membrane-Bound Guanylyl Cyclase

鸟苷酸环化酶以可溶和/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，细胞中cGMP浓度的增加被认为是多种因素的结果。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)。纯化后的131kDa和128kDa鸟酰环化酶含26.0sy。-.])1.3和4.3(]sy.-.])每摩尔蛋白分别为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)。

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).