A mRNA for Membrane Form of Guanylyl Cyclase ls Expressed Exclusively in the Testis of the Sea Urchin Hemicentrotus pulcherrimus Takeshi Shimizu

A cDNA cione encoding the membrane form of guanylyl cyclase was isolated from a Hemicentrotus pulcherrT'mus testis cDNA library and its nucleotide sequence was determined. The cDNA was 4123 bp long and an open reading frame predicted a protein ot 1125 amino acids including an apparent signal peptide of 21 residues; a single transmembrane domain of 25 amino acids which dMdes the mature protein into an amino-terminal, extracellular domain of 485 amino acids and a carboxyl-terminal, intracellular domain of 594 amino acids. Three potential N-linked glycosylation sites were present in the extracellular domain, Northern blot analysis of poly(A}'RNA from te$tes, ovaries, eggs and embryos at various developmental stages showed that the cDNA encoding guanylyl cyclase hybridized to a mRNA of 4.4 kb from the testes. We developed a large scale purification method of the phosphorylated {131 kDa) and dephosphorylated (128 kDa} forms of the membrane-bound guanylyl cyclase from H, pulcherrimus spermatozoa. The purified 131 kDa and 128 kDa forms of the guanylyl cyciase contained 26.0 ± 1.3 and 4.3 ± O.7 moles of phosphate per mol protein (mean ± S.D.: n=6}, respectively. The amino-terminal amino acids of both the 131 kDa and 128 kDa forms of the guanylyl cyclase could not be detected, suggesting that they were blocked.