Study on Phosphorylating Sites of Membrane-Bound Guanylyl Cyclase

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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).