Study on Phosphorylating Sites of Membrane-Bound Guanylyl Cyclase

关酰环化酶以可溶性和/或颗粒形式存在于各种细胞隔间中，并催化GTP形成cGMP和无机焦磷酸盐。长期以来，人们已经知道细胞中的cGMP浓度会增加到各种各样的药物。海胆精子中cGMP浓度的短暂增加可以解释为guanyyl环化酶的短暂激活和随后的失活，这与酶的磷酸化状态密切相关。因此，确定关酰环化酶的磷酸化位点是很重要的。在本项目中，我们从海胆hemicentrus pulcherrimus精子中开发了一种大规模纯化磷酸化（131kDa）和去磷酸化（128kDa）形式的膜结合鸟酰环化酶的方法。纯化的131kDa和128kda形式的胍基环化酶每摩尔蛋白质分别含有26.0(]SY.+-.])1.3和4.3(]SY.+-.])0.7摩尔磷酸。我们从131kDa形式的关尼酰环化酶的赖基内多肽酶消化中分离出两个磷酸化肽。一个（GSLQDILENDDIK）对应于651 - 663（激酶样结构域），另一个（TEQLLHRMLPPSIASQLIK）对应于883 - 901（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).