Immunohistochemical investigation of gamma-aminobutyric acid ontogeny and transient expression in the central nervous system of Xenopus laevis tadpoles

A cDNA clone encoding the membrane form of guanylyl cyclase was isolated from a Hemicentrotus pulcherrimus testis cDNA library and its nucleotide sequence was determined. The cDNA was 4123 bp long and an open reading frame predicted a protein of 1125 amino acids including an apparent signal peptide of 21 residues; a single transmembrane domain of 25 amino acids which divides 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 testes, 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 cyclase contained 26.0 +/- 1.3 and 4.3 +/- 0.7 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.