A mRNA for membrane form of guanylyl cyclase is expressed exclusively in the testis of the sea urchin Hemicentrotus pulcherrimus.
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.
Shimizu
T
T
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
Takeda
K
K
Furuya
H
H
Hoshino
K
K
Nomura
K
K
Suzuki
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D006162:Guanylate Cyclase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism*; D012617:Sea Urchins; D017422:Sequence Analysis, DNA; D013737:Testis / Q000201:enzymology*
D018076:DNA, Complementary; D012333:RNA, Messenger; D006162:Guanylate Cyclase