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The mRNA for component C1 of rat prostatic binding protein has been cloned and characterized. A partially purified mRNA fraction for this complex protein was reverse-transcribed into double-stranded cDNA and cloned into the PstI site of plasmid pBR 322. The 426-base-pair insert of the recombinant plasmid pC1A75 was completely sequenced. The coding region corresponds precisely to the 88 amino acid residues of C1 and in addition contains the information of a signal peptide of 23 residues. The 5' non-coding region counts only 19 nucleotides and is incomplete but the 3'-terminal non-coding part of 60 nucleotides extends into the poly(A) tail. Sequence analysis of other C1-positive clones indicates the presence of sequence rearrangements which must have occurred during the cloning procedure. Possible mechanisms for the generation of these cloning artefacts are discussed.

The prostatic binding protein, previously described in rat ventral prostate, was isolated. The purified protein binds pregnenolone with an affinity of 1.2 X 10(6) M-1 and contains an average of 0.84 binding site per molecular. Its carbohydrate content is 3.2%. Its Mr, estimated by gel filtration, is 51 000 but in the presence of 6 M guanidine hydrochloride or 0.1% dodecylsulfate it dissociates into two subunits (S and F), which can be separated by polyacrylamide gel electrophoresis or by chromatography on hydroxyapatite. The Mr of these subunits is about 17 000, when estimated by gel filtration in 6 M guanidine hydrochloride, or 19 000 for subunit F and 20 000 for subunit S, when measured by dodecylsulfate/polyacrylamide gel electrophoresis. Their isoelectric points, estimated by isoelectric focusing in 8 M urea, are 4.6 for subunit F and 4.9 for subunit S. Prostatic binding protein and both subunits have a very similar amino acid composition. Upon reduction of disulfide bridges each subunit dissociates further into two components: one of these components is the same in both subunits.

The amino acid sequence of component C2, the polypeptide specific for subunit S of prostatic binding protein, the major secretory glycoprotein of the rat ventral prostate, has been determined. Its structure was established using the manual Edman degradation on the most relevant fragments obtained by enzymatic digestion of the S-carboxamidomethylated component C2 and the native subunit S and by chemical cleavage of the remaining undigestible 'cores' with cyanogen bromide. Component C2 contains 92 amino acids corresponding to a molecular weight of 10619. It is a slightly acidic polypeptide in which the acidic and basic residues are unevenly distributed. The N terminus is blocked and three cysteine residues are almost evenly distributed over the peptide chain. A highly polar region is found in position 23-34 and two hydrophobic segments are located in the C-terminal part of the molecule. Component C2 is compared with component C1 of subunit F and their high sequence homology reveals an evolutionary relationship.

The proline-rich polypeptide, that is bound to rat prostatic binding protein displays a marked heterogeneity on isoelectric focusing, with major bands at pH 7.6 and pH 6.9. The same complex pattern is obtained for PRP prepared from prostates of individual rats from several strains. Using carboxymethylcellulose chromatography 6 different forms of PRP can be separated. Five of them have the same size (MW : 4000) and respectively glycine and lysine as N- and C-terminal amino acid. Their amino acid composition suggests that these forms differ by internal substitution respectively of aspartic acid and glycine and of proline and histidine. The sixth form (MW : 3500) lacks several amino acids at its N-terminal.

The amino acid sequence of the glycosylated component C3 of rat prostatic binding protein has been determined. The peptides obtained by digestion of the S-carboxamidomethylated or S-aminoethylated glycoprotein with trypsin and Staphylococcus aureus protease were sequenced by manual Edman degradation. The alignment of the fragments was further established with overlapping peptides obtained by enzymic hydrolysis of the modified protein with chymotrypsin and thermolysin, and by chemical cleavage with cyanogen bromide. The glycopeptide C3 contains 77 amino acids corresponding to a molecular weight of 8653. the oligosaccharide chain is attached to the peptide by an N-glycosidic bond to asparagine-17. C3 is an acidic polypeptide due to the presence of ten acidic residues; its three cysteine residues are located at both extremities and in the middle of the molecule.

A proline-rich polypeptide is associated with prostatic binding protein, a major androgen-dependent protein described previously in the rat ventral prostate. This polypeptide has been purified. Its molecular weight estimated by gel filtration is about 8500, but a markedly lower value (3300) is obtained by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. Isoelectric focusing on thin layer polyacrylamide gels yields two major forms with isoelectric points of, respectively, 7.75 and 7.05. The amino acid composition of proline-rich polypeptide is characterized by a high (19.5%) proline content and its NH2-terminal amino acid is glycine. Like prostatic binding protein, proline-rich polypeptide is a characteristic component of the rat ventral prostate and localized primarily in the intraluminal secretion of this gland. In intact adult male rats the cytosol of a whole gland contains 0.70 +/- 0.15 (S.D.) mg of the polypeptide, as measured by radial immunodiffusion or 2.6 +/- 0.5% of (S.D.) of the total protein. This amount decreases gradually after castration and becomes undetectable after 8 days. Androgen treatment, on the other hand, results in a rapid stimulation, while estradiol and progesterone are ineffective. Proline-rich polypeptide is markedly more androgen-dependent than prostatic binding protein, and promises to be an interesting end point for studies on the mechanism of action of androgens.

The complete primary structures of the two main forms, PRP-IV and PRP-V, of a proline-rich polypeptide bound in vivo to rat prostatic binding protein has been determined. Their sequences were established using manual Edman degradation of the native polypeptide and of purified fragments derived from trypsin and thermolysin digestions. Both polypeptides contain 38 amino acid residues (Mr = 4397 and 4339); cysteine, methionine, and serine are missing. In spite of the high proline content (21%), no polyproline stretches were detected. PRP-IV and PRP-V show an extensive structural homology and differ only by three substitutions. These amino acid replacements are located in the NH2-terminal part of the molecule at positions 6 (His leads to Pro), 10 (Pro leads to His), and 11 (Asp leads to Gly). Moreover, each component displays a microheterogeneity at several positions in the sequence which indicates that multiple structural variants exist for PRP-IV and PRP-V. These data not only suggest the existence in rat ventral prostate of a multigene family coding for the proline-rich polypeptides but also the occurrence of a pronounced genetic polymorphism for these components. In addition, a remarkable sequence homology is observed between the PRP components and the region of the B chain in the precursor of mouse renin.

The amino acid sequence of component C1, the polypeptide specific for subunit F of prostatic binding protein, the major secretory glycoprotein of the rat ventral prostate, has been determined. Its structure was established using the manual Edman degradation on the intact protein and on the most relevant fragments isolated from trypsin, chymotrypsin, thermolysin and Staphylococcus aureus protease digests of the 14C-labelled S-carboxamidomethylated component C1. Component C1 contains 88 amino acids corresponding to a molecular weight of 10246. It is an acidic polypeptide due to the presence of 17 acidic residues; its three cysteine residues are almost symmetrically distributed over the peptide chain. Highly polar regions are found in positions 17-27 and 37-47, while the C-terminal part of the molecule contains two hydrophobic segments.