CDNA CLONING IDENTIFIED A CALMODULIN-BINDING PROTEIN IN BOVINE SEMINAL PLASMA AS BOVINE C-TYPE NATRIURETIC PEPTIDE

A basic protein of apparent molecular weight 15 kD, designated bSVSP15 , was purified from bovine seminal vesicle secretion to homogeneity, e mploying affinity absorption to calmodulin-Sepharose and reverse-phase HPLC. Immunoblotting identified bSVSP15 in bovine seminal plasma and seminal vesicle secretion, but it was not present either in extracts o f bovine ampulla, epididymis, and testis or in serum or follicular flu id. When added to cAMP phosphodiesterase, bSVSP15 inhibited the activa tion of enzymatic activity by calmodulin in a reversible manner. Immun oscreening of a lambda gt11 expression cDNA library from bovine semina l vesicle tissue yielded two positive clones, pSVS4 and pSVS5, which w ere characterized by sequencing. Both sequences are identical, except for the 3' region. Because the derived amino acid sequence comprises, with an identity of 81%, the amino-terminal 21 residues of bSVSP15, cD NA clones pSVS4 and bSVS5 represent bSVSP15-specific mRNAs. The mature protein bSVSP15 contains 101 residues and is preceded by 25 residues of a signal sequence, characteristic for secretory proteins. Northern analysis identified two bSVSP15-specific mRNAs of 900 bp and 1200 bp, respectively. Sequence comparison yielded high homologies to human C-t ype natriuretic peptide. We conclude from this result that bSVSP15 is identical with the hitherto unknown bovine C-type natriuretic peptide.