Calcium-binding protein in bull seminal vesicle secretion and seminal plasma.

A protein which showed high affinity for calcium ions was isolated from bull seminal vesicle secretion and seminal plasma. Its calcium-binding activity depended on the ionic strength and pH of the medium. The dissociation constant was 7-7 X 10(-7) M and there were 14 binding sites per protein molecule. The molecular weight of calcium-binding protein from bull seminal vesicle secretion, estimated by the gel filtration method, was 110,000. The protein may be involved in the regulation of the calcium ion level in seminal plasma.     

The major protein of bull seminal plasma is a secretory product of seminal vesicle

We isolated the major protein with apparent molecular weight, Mr, 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as in seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid, respectively. Cell-free translation of poly(A+)RNA isolated from seminal vesicle tissue resulted in formation of one major species with apparent Mr 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. We thus provided evidence that the major protein component of bull seminal plasma is a secretory protein of seminal vesicles. Furthermore, it appeared that the isolated major protein may be closely related to the protein PDC109, purified from bull seminal plasma and sequenced by Esch et al. (Biochem. Biophys. Res. Commun. 113, 861-867 (1983).     

Characterization of basic proteins of bull seminal plasma

We have employed high-performance liquid chromatography on reversed phase columns to analyse the major basic proteins from bull seminal plasma. The proteins were separated preparatively and characterized with respect to molecular mass, amino-acid composition as well as by means of immunodiffusion against specific antisera. The following proteins could be identified: bull seminal proteinase inhibitor II (BUSI II), two seminal RNAases, the seminal antimicrobial protein and proteolytic fragments, derived from it, and a hitherto unknown protein P6 of molecular mass 20 000 Da. Another unknown protein, P5, found to be formed during preparation of the basic protein fraction turned out to be a proteolytic fragment of protein P6 with a molecular mass of 8 750 Da for the polypeptide chain. Antisera against the isolated proteins were raised in rabbits and their specificity established. Single radial immunodiffusion was used to determine the concentration of the above basic proteins in bull seminal plasma: BUSI II (0.25 mg/ml), seminal RNAases (6.5 mg/ml) and protein P6 (2.9 mg/ml).     

Stimulation of bull seminal RNase by various basic proteins

The activity of purified bull seminal RNase was markedly stimulated by various basic proteins. At the half concentration of substrate RNA, basic proteins such as histones, high-mobility group chromosomal proteins and cytochrome c stimulated the enzyme activity 4-6 fold. Other non-basic proteins such as bovine serum albumin and human gamma-globulin were far less effective. In addition to enzyme-stimulating activity, basic proteins showed a marked enzyme-stabilizing activity, indicating the presence of a strong interaction between the enzyme and basic proteins.     

Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle.

1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug.     

Seminalplasmin,the major basic protein of bull seminal plasma,is a secretory protein of the seminal vesicles

From the experimental results of three independent methods: (1) indirect immunofluorescence employing monospecific anti-seminalplasmin-IgGs, (2) cell-free translation of poly(A)⁺ RNA from seminal vesicle and testicular tissue, as well as (3) Northern analysis of poly(A)⁺ RNA of the latter tissues with a synthetic seminalplasmin-specific antisense DNA probe, it is concluded that the biosynthesis of seminalplasmin occurs in seminal vesicles but not in testis.     

Alanyl aminopeptidase of bovine seminal vesicle secretion

Multiple forms of an aminopeptidase hydrolysing L-alanine- and various other amino acid-beta-naphthylamides in bovine seminal vesicle secretion were studied after fractionation on gel filtration, anion exchange chromatography and chromatofocusing. Two forms of the enzyme were found in all these fractionations: one with a high molecular weight was aggregated or particle-bound and the other had a molecular weight of about 237,000. The high-molecular-weight form dissociated with Triton X-100 via an intermediate into the basic enzyme form with concurrent change in the pI and anionic sites. The basic form of the enzyme differed from the high-molecular-weight forms in substrate preference, response to some modifiers, thermal stability and kinetic constants.     

Phosphorylation of sperm-binding proteins from seminal vesicle secretion by sperm surface protein kinase

Rat seminal vesicle secretion does not demonstrate protein kinase activity, either towards endogenous or exogenous proteins. When epididymal sperm were incubated in vitro with seminal vesicle secretion, three prominent secretory proteins were bound to the sperm. Two of these proteins were highly phosphorylated. Thus, selected sperm-binding proteins from accessory gland secretions are phosphorylated by sperm surface protein kinases.     

Interaction of proteins RSV IV and RSV V in rat seminal vesicle secretion

The RSV IV polypeptide, molecular weight ratio (Mr = 10,000), which is produced by the rat seminal vesicle, has previously been suggested to be associated with another polypeptide in the gland secretion (Higgins et al., '76). This study provides that RSV IV is a component of a protein shown by immunoassays, electrophoresis, and amino acid composition analysis to contain, together with RSV IV, the seminal vesicle secretory RSV V polypeptide (Mr = 13,000). This RSV IV-RSV V complex (namely CFS protein) had an isoelectric point at pH 7.2 and an approximate molecular weight of 22,000 daltons. This complex inhibits the previously reported in vitro binding of the isolated RSV IV to epididymal sperm cells, thus suggesting a functional role for the RSV IV-RSV V interaction.     

Seminalplasmin, the major basic protein of bull seminal plasma, is a secretory protein of the seminal vesicles.

From the experimental results of three independent methods: (1) indirect immunofluorescence employing monospecific anti-seminalplasmin-IgGs, (2) cell-free translation of poly(A)+ RNA from seminal vesicle and testicular tissue, as well as (3) Northern analysis of poly(A)+ RNA of the latter tissues with a synthetic seminalplasmin-specific antisense DNA probe, it is concluded that the biosynthesis of seminalplasmin occurs in seminal vesicles but not in testis.     

Affinity chromatography of bull seminal proteins on mannan-Sepharose

4-Hydroxynon-2-enal (4-HNE) is one of the major aldehydic products of lipid peroxidation (LPO) and is involved in a number of pathophysiological processes. Since LPO products are useful indicators for oxidative stress in vivo, a number of detection methods for LPO products in biological tissues were developed. However, none of these methods is presently used in clinical settings. In order to introduce LPO products as biomarkers in clinical studies a suitable GC-MS method for 4-HNE detection was adapted to meet clinical requirements. As one result, the minimal sample volume could be decreased to 50 microl of plasma so that the method might even be suitable for pediatric purposes. The best internal standard (I.S.) for 4-HNE detection by GC-MS 9,9,9-D(3)-4-hydroxynon-2-enal was introduced by van Kuijk et al. [Anal. Biochem., 224 (1995) 420]. However, because of its limited availability, benzaldehyde-ring-d(5), 4-hydroxybenzaldehyde, and 2,5-dihydroxybenzaldehyde were tested to find an alternative. Out of these three, 4-hydroxybenzaldehyde was shown to serve best as I.S. To examine the applicability of the adapted method, tests on the stability of 4-HNE in samples during storage were carried out. It was shown that plasma samples need to be stored at -80 degrees C or less to avoid greater loss of 4-HNE. Samples with 4-HNE concentrations close to the physiological level were shown to be stable over 22 months at -80 degrees C. The introduction of a new and easily available I.S., reduction of the sample volume, and information about sample stability provided by this study facilitate 4-HNE determination in most clinical settings.     

The major protein of bull seminal plasma: Biosynthesis and biological function

We isolated the major protein of apparent M_r of 15,000–16,000 from seminal plasma as well as from seminal veiscle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid respectively.Cell-free translation of poly(A)RNA from seminal vesicle tissue and immunoprecipitation yielded one major species with apparent M_r of 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. Cloning and sequencing of a major protein-specific cDNA led to the identification of clone pMP17, encoding a precursor of the major protein of 128 amino acid residues. We proved that the major protein is identical to protein PDC 109 (Eschet al., Biochem. Biophys. Res. Comm. 113:861–867, 1983).The seminal vesicles synthesize major protein in an androgen-dependent fashion. In addition to intraluminal secretion of the vas deferens, ampullary spermatozoa revealed an intense immunoreaction which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Epididymal epithelium (as well as calf seminal vesicle epithelium) showed no immunoreactivity with major protein antiserum. Immunoelectron microscopy demonstrated that only spermatozoa devoid of a plasma membrane around the middle piece were able to bind the antiserum against major protein. After removal of the plasma membrane from epididymal spermatozoa, binding of major protein to subplasmalemmal binding sites was visualised using gold-labeled MP.Transblotting with gold-labeled MP demonstrated a protein of about 66 kDa which appears to represent the major protein-receptor. Binding of major protein to the receptor (after loss of the plasma membrane in the mid-piece region of the spermatozoa after contact with secretions from seminal vesicles) is interpreted as a phyisological process presumably related to the onset of sperm motility.