Concanavalin A induced inhibition of 5'-nucleotidase from guinea pig skeletal muscle and bull seminal plasma: a comparative study

Both purified and membrane-bound 5'-nucleotidases (EC 3.1,3.5) from guinea pig skeletal muscle and bull seminal plasma are inhibited by Concanavalin A (Con A). 5'-Nucleotidase purified from skeletal muscle is inhibited by Con A by an apparent uncompetitive process (K'i = 160 nM), while the lectin inhibits the particulate enzyme by an apparent non-competitive process (Ki = K'i = 50 nM). 5'-Nucleotidase purified from bull seminal plasma is inhibited by Con A by an apparent non-competitive process (K'i = Ki = 270 nM), while the membrane-bound enzyme is subjected to a mixed type inhibition by the lectin (K'i greater than Ki; 30 and 14 nM, respectively). The enzyme purified from skeletal muscle exhibits a significant cooperativity in the interaction with Con A. The inhibition of bull seminal plasma particulate 5'-nucleotidase brought about by Con A is not completely reversed by addition of alpha-methyl-D-mannoside.     

Purification of 5'-nucleotidase from human seminal plasma.

5'-Nucleotidase from human seminal plasma was purified to electrophoretic homogeneity and some of its kinetic and molecular properties compared with those of 5'-nucleotidase from bull seminal plasma. The purification of the enzyme was achieved by using the same affinity chromatography media (Con A-Sepharose and AMP-Agarose or ADP-Agarose) previously used for the purification of bull seminal plasma 5'-nucleotidase (Fini, C., Ipata, P.L., Palmerini, C.A. and Floridi, A. (1983) Biochim. Biophys. Acta 748, 405-412). However, in the present purification procedure no detergent was used as it had been necessary for the purification of the bovine enzyme. The experimental data reveal some main differences between these two enzymes; first, the human enzyme seems to be constituted of a single polypeptide chain of about 71 kDa, while the 5'-nucleotidase of bull seminal plasma, in non denaturing detergent solutions, is a homodimer of about 160 kDa. Another most remarkable difference is that the human enzyme does not seem to contain a phosphatidylinositol anchoring system like the one present in the bovine enzyme and in 5'-nucleotidase of different sources (Low, M.G. (1987) Biochem. J. 244, 1-13). Finally, the AMPase activity of 5'-nucleotidase from human seminal plasma is not affected by dithiothreitol which, on the contrary, is a powerful inhibitor of the bovine enzyme causing the dissociation of its subunits which are held together by disulphide bridges (Fini, C., Minelli, A., Camici, M. and Floridi, A. (1985) Biochem. Biophys. Acta 827, 403-409).     

beta-Glucuronidase in the bull seminal plasma and reproductive organs

The biochemical distribution of beta-glucuronidase activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity was found in the epididymis, where the activity seemed to be mostly in nonsecretory and only partly in secretory form. A molecular weight of 340 X 10(3) to 360 X 10(3) was recorded for beta-glucuronidase in the bull seminal plasma and different reproductive organs with gel filtration on Sepharose 6B. In chromatofocusing four activity areas (CF-1 to CF-4) were usually obtained for beta-glucuronidase in the bull seminal plasma. The major peak CF-2 (also in the different reproductive organs) had a pI value of 5.6-5.3 and the two minor activity areas CF-1 and CF-3 had pI values of 6.0-5.8 and 5.2-4.5, respectively. Peak CF-4 eluted with a NaCl gradient after the Polybuffer elution and possibly represents an enzyme form incompletely detached from negatively charged cellular material. Isoelectric focusing on polyacrylamide gel confirmed the heterogeneity of beta-glucuronidase, since several activity bands were detected in the secretion of the different parts of the epididymis. beta-Glucuronidase activities CF-1, CF-2 and CF-3 had similar pH activity profiles (pH optimum around pH 3.0-4.0) and response to thermal inactivation at 50 degrees C. The multiple beta-glucuronidase activities of the bull seminal plasma are proposed to derive mainly from the secretion of the cauda epididymidis.     

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).     

Acid and neutral alpha-glucosidase in the reproductive organs and seminal plasma of the bull

A synthetic substrate (p-nitrophenyl-alpha-D-glucopyranoside) was used to measure the acid and neutral alpha-glucosidase activity in bull seminal plasma, spermatozoa and in homogenates of bull reproductive organs. Marked differences were observed in the activities of these enzymes in the various tissues studied. Epididymis and particularly its caput region contained the highest specific activity of acid alpha-glucosidase. The activity of neutral alpha-glucosidase was highest in testis and in different parts of the epididymis. Seminal plasma, spermatozoa and seminal vesicle secretion contained only the acid enzyme activity. After fractionation with anion exchange chromatography in HPLC (Mono Q) and chromatofocussing, acid alpha-glucosidase activity of seminal plasma was recovered in two fractions with different pI values. The corresponding activities were found in the secretion of seminal vesicles, which thus form the major secretory source of seminal plasma acid alpha-glucosidase. In the fractionation with gel filtration on Sepharose 6B, the acid alpha-glucosidase had a smaller molecular weight than did the neutral enzyme. In anion exchange chromatography and chromatofocussing the testicular and epididymal homogenates each contained two acid and two neutral isoenzymes. In both fractionations the elution pattern of acid alpha-glucosidase was clearly different from that of the enzymes in seminal plasma. The pH optimum of acid alpha-glucosidase ranged from 3.75 to 4.5 and that of the neutral enzyme from 6.5 to 7.0. The neutral activity was more sensitive to many divalent metal ions and differences were also observed in the response of the enzymes to different concentrations of turanose and KCl.     

Selenium and glutathione peroxidase in seminal plasma of men and bulls

High levels of selenium and glutathione peroxidase (GSH-Px) were found in bull seminal plasma but low concentrations in human seminal plasma. In man the seminal plasma selenium was associated with two macromolecules separable by gel filtration, but no GSH-Px was found in the same fractions. Selenium in bull seminal plasma was associated with two proteins, which could be separated by gel filtration and anion exchange chromatography. Both macromolecules coeluted with GSH-Px activity and had identical optima at pH 7.0. Their responses to thermal treatment, however, differed. Seminal vesicle secretory fluid in the bull contained both these proteins, while the larger molecule was also found in fractionations of ampulla, prostate and Cowper's glands. The larger enzyme form is evidently a tetramer of the smaller one. Both enzyme forms were extremely sensitive to heavy metals and some divalent metal ions. GSH caused an activation while other reducing agents were suppressive. Triton X-100 had no effect, while sodium deoxycholate was inhibitory. These properties are typical for a phospholipid hydroperoxide GSH-Px. It is concluded that this selenium-dependent enzyme may be important in the protection of bovine spermatozoa against damage caused by oxygen radicals, while in man such a mechanism is not functional.     

α-L-fucosidase in the reproductive organs and seminal plasma of the bull

α-L-Fucosidase (EC 3.2.1.51) activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of α-L-fucosidase was found in the epididymis. Gel filtration at pH 7.0 revealed two α-L-fucosidases (α-L-fucosidase I and α-L-fucosidase II) in most reproductive tissues, but seminal plasma, spermatozoa and epididymal cauda contained only form I. Fractionation at basic pH (pH 8.5) resulted in the elution of α-L-fucosidase as form II. Some differences were encountered in pH profiles and thermal stabilities of the two enzyme forms and they showed additional polymorphism after chromatofocusing. The comparison of enzyme profiles after fractionations suggests that cauda epididymidis is the main source of the seminal plasma activity in the bull.     

Characterization of acid and neutral alpha-mannosidases in bull semen and reproductive organs

Acid and neutral alpha-mannosidase activities were studied in the bull reproductive tissues, isolated spermatozoa, epididymal and seminal vesicle secretion and seminal plasma. The acid enzyme in the seminal plasma mainly derived from the epididymal secretion, while the neutral one was enriched in the sperm cells. The latter activity in the seminal plasma appears to be due to an enzyme released from the cytoplasmic droplets in the epididymis. The acid enzyme had a molecular weight of 220,000-320,000, pI 7.3-6.0 and an optimum at pH 4.0. It was sensitive to swainsonine but was stimulated by Zn2+. The neutral enzyme had a molecular weight of 360,000-460,000, pI 5.4-4.7 and showed double optima at pH 5.5 and 6.0-7.0. It was resistant to swainsonine but was markedly activated by Co2+ or Fe2+. The neutral enzyme was also more sensitive to thermal inactivation than the acid one.     

Beta-galactosidase in the seminal plasma and reproductive organs of the bull

The distribution of beta-galactosidase activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of beta-galactosidase was found in testis and in different parts of the epididymis, where the activity seemed to be partly in secretory (cauda secretion) and partly in non-secretory, bound form (caput to cauda epididymidis). Gel filtration on Sepharose 6B at pH 7.0 revealed two beta-galactosidase forms (GF-1, Mr approximately 500,000-600,000 and GF-2, Mr approximately 190,000-220,000) in reproductive organs and seminal plasma. The pH-optimum of both beta-galactosidase forms was about 3.75-4.75. Hg2+ and p-chloromercuribenzoate inhibited strongly these activities. Further, form GF-2 seemed to be slightly more sensitive to the thermal inactivation at 50-70 degrees C than form GF-1. In chromatofocusing beta-galactosidase activities in bull seminal plasma coeluted with those of the cauda epididymidis (pI-values 7.5-6.4). On the contrary, prostate, Cowper's gland, testis, ampulla and seminal vesicles had enzyme activities eluting at lower pI-values (6.3-4.2). Thus, the seminal plasma activity is mainly an indicator for the function of the epididymal cauda.     

alpha-L-Fucosidase in the reproductive organs and seminal plasma of the bull

alpha-L-Fucosidase (EC 3.2.1.51) activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of alpha-L-fucosidase was found in the epididymis. Gel filtration at pH 7.0 revealed two alpha-L-fucosidases (alpha-L-fucosidase I and alpha-L-fucosidase II) in most reproductive tissues, but seminal plasma, spermatozoa and epididymal cauda contained only form I. Fractionation at basic pH (pH 8.5) resulted in the elution of alpha-L-fucosidase as form II. Some differences were encountered in pH profiles and thermal stabilities of the two enzyme forms and they showed additional polymorphism after chromatofocusing. The comparison of enzyme profiles after fractionations suggests that cauda epididymidis is the main source of the seminal plasma activity in the bull.     

gamma-Glutamyl transpeptidase of human seminal fluid.

γ-Glutamyl transpeptidase, which is present in high levels in human seminal fluid plasma, was purified about 870-fold from this source. The enzyme is present in seminal fluid plasma in particulate form. Purification by a procedure involving treatment with bromelain gave a protein (apparent molecular weight, about 70,000), which exhibited catalytic properties characteristic of γ-glutamyl transpeptidase preparations isolated from rat kidney and other mammalian tissues. The physiological significance of seminal fluid γ-glutamyl transpeptidase and its potential clinical value are considered.     

Sphingomyelinases in human, bovine and porcine seminal plasma

The seminal plasma of man, boar and bull was found to have a sphingomyelinase (SMase) activity hydrolysing [N-methyl-14C]sphingomyelin. The human and porcine enzymes had an acid pH optimum and were not influenced by divalent metal ions or chelating agents. They were closely similar with the lysosomal enzyme in many tissues. The bovine seminal plasma SMase was partially purified. The enzyme was a glycoprotein with pH optimum at 6.5, a broad pI 4.2-4.8 and molecular mass of 160 and 60 kDa, respectively, in native and SDS-PAGE. The enzyme was activated by Co greater than Mn greater than Cd greater than Ni and inhibited by chelating agents, Cu, Fe, Pb and Zn. The enzyme was clearly distinct from the acid lysosomal SMase and the previously described neutral Mg2+-dependent and independent activities. It had a wide distribution in the bull reproductive tissues.     

Immunohistochemical localization of phospholipase A2 in the bovine seminal vesicle and on the surface of the ejaculated spermatozoa.

1. Approximately 150-fold purified phospholipase A2 (PLA2) from bovine seminal vesicle fluid was injected into rabbit to prepare antibodies. 2. Produced antisera blocked PLA2 activity in bovine seminal plasma, seminal vesicles and its fluid and it gave single precipitation lines with the same samples. No cross-reactivity was detected with other reproductive tissues of bull as well as human seminal plasma. 3. Using indirect peroxidase technique PLA2 was localized in the apical part of epithelia cells of the bull seminal vesicle and also some minor immunohistochemical reactions were observed in the tubular lumen. Indirect peroxidase staining gave weak or no reaction at all to seminal vesicles of immature bulls. This suggests that the enzyme may be under hormonal control. 4. By indirect immunofluorescence method ejaculated spermatozoa of bull revealed immunoreaction which was not uniform and it was restricted to the middle piece, acrosome as well as postacrosomal region, but no specific immunostaining could be found on the surface of the epididymal spermatozoa. 5. Enzyme visualization by immunoelectron microscopic labelling showed a predominant localization in membrane particles inside the lumen of bovine seminal vesicle but some gold particles were also seen in granules, larger vacuoles and in cytoplasm of epithelia cells.     

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).     

Analysis of lipid and protein components of ejaculated bull sperm surface and seminal plasma

Surface components of ejaculated bull sperm were radiolabeled by enzymatic iodination with lactoperoxidase and Na¹²⁵I. The sperm were lysed and the labeled components analyzed on SDS-7.5% polyacrylamide gels. Electrophoresis of solubilized radioactivity resolved six components with approximate molecular weights of 77, 61, 44, 36, 24, and 15 kilodaltons. To identify components that might be adsorbed to the bull sperm surface from seminal secretions, seminal plasma was labeled. Electrophoresis of labeled seminal plasma resolved four components with approximate molecular weights of 74, 33, 24, and 15 kilodaltons, each of which comigrated with a labeled sperm surface component. To identify the chemical composition of the radiolabeled components, labeled sperm surface and labeled seminal plasma were submitted to isopycnic density gradient centrifugation in cesium chloride. The ¹²⁵I incorporated into bull sperm surface separated into two discrete areas of radioactivity, one having a density characteristic of protein and the other, of lipid. Iodinated seminal plasma banded in one discrete area that had a density characteristic of protein. Electrophoretic analysis of each area of radioactivity recovered from the gradients demonstrated that five of the six sperm surface and all of the seminal plasma components were in the protein fractions. The 15-kilodalton sperm surface component banded as a lipid, whereas the 15-kilodalton seminal plasma componènt banded as a protein.     

Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process

Experiments were conducted to investigate the effect of seminal plasma on sperm motility during the cryopreservation process. Ejaculated and epididymal spermatozoa from the ram and the bull were washed by centrifugation and resuspended in either seminal plasma or a modified Tyrode's medium (TALP) prior to dilution in medium suitable for cryopreservation. Resuspension of washed ejaculated ram spermatozoa in seminal plasma resulted in higher percentages of motile spermatozoa than resuspension in TALP after the spermatozoa were cooled to 5 degrees C (52 vs 35%), and after thawing (14 vs 9%), respectively. Resuspension of epididymal ram spermatozoa in seminal plasma had no beneficial effect in maintaining sperm motility after cooling (78 vs 73%); however, seminal plasma was beneficial to epididymal ram spermatozoa after thawing (34 vs 3%), respectively. Resuspension of washed ejaculated bull spermatozoa in either seminal plasma or TALP had no effect on the percentage of motile spermatozoa after cooling to 5 degrees C (73 vs 75%) or after thawing (60 vs 60%), respectively. In addition, seminal plasma had no beneficial effect on the percentage of motile epididymal bull spermatozoa when compared with that of TALP-treated spermatozoa after cooling (75 vs 72%) or after thawing (66 vs 63%), respectively. Seminal plasma from different sires (ram and bull) affected epididymal sperm motility. The ability of sperm cells to withstand damage during cryopreservation, however, appears to reside in the sperm cells themselves, probably due to sperm cell composition.     

The major basic proteins of bull seminal vesicle secretion

We have employed HPLC on reversed phase columns to analyse the major basic proteins from bull seminal vesicle secretion. The identification of proteins was achieved by comparison with authentic protein samples from bull seminal plasma as well as immunological characterisation using antisera directed against the latter proteins. The major basic proteins from bull seminal plasma: bull seminal proteinase inhibitor II (BUSI II), the seminal ribonuclease BS1, the protein P6 as well as the antimicrobial protein were also identified as the main constituents of the fraction of basic proteins derived from seminal vesicle secretion. FPLC using Mono S HR columns was also found to resolve the mixture of basic proteins and proved to be especially useful with respect to the isolation of the antimicrobial protein from basic proteins of seminal vesicle secretion. The identity of the antimicrobial protein from bull seminal plasma with the respective protein from seminal vesicle secretion was confirmed by amino-acid analysis and comparison of tryptic peptide patterns by HPLC. The antimicrobial protein was isolated from seminal vesicle secretion with a yield of 3 mg/ml of secretion.     

DNA-dependent protein methylase activity in bull seminal plasma.

The existence of a DNA-dependent protein methylase activity without any concomitant DNA methylase activity was demonstrated in bull seminal plasma. The enzyme utilized S-adenosyl-L-methionine as a methyl donor, and endogenous seminal plasma protein as the substrate. There was no demonstrable enzyme activity when the seminal plasma was preheated at 100 degrees for 10 min, or when the enzyme reaction mixture was incubated at 4 degrees. The protein methylase required a heterologous DNA source, had optimal activity at pH 8.1, and was enhanced in the presence of Mg2+, NH4+, and reduced glutathione. After the methylated protein product was separated from DNA by extraction with 0.2 M HCl, the incorporated radioactivity was shown to be totally solubilized by incubating the protein either with Pronase or 1 M NaOH, while RNase and DNase had no effect. Approximately 70% of the enzymatically synthesized amino acids in the protein product were tentatively identified as O-methylated amino acid ethers by virtue of their elution from a Dowex 50 H+ column with 0.2 M pyridine, and their stability to acid and base hydrolysis. The partially purified methylated product was shown by Sephadex G-50 chromatography to consist of three distinct radioactive proteins with molecular weights of approximately 21,000, 15,000, and 10,000.     

Adenosine metabolizing enzymes in seminal plasma of bull and man: a comparative study

1. Adenosine metabolizing enzymes in seminal plasma of man and bull have been investigated. 2. A different level of 5'-nucleotidase activity has been found in two seminal plasmas: in bull 5'-nucleotidase represents 80% of the total AMP dephosphorylating enzymes while in man 5'-nucleotidase represents only 1.3% of the total AMP dephosphorylating activities. 3. Apart from the different levels of 5'-nucleotidase activity, different kinetic parameters have been reported for 5'-nucleotidase, acid prostatic phosphatases, ADA and PNP. 4. Adenosine kinase, xanthine oxidase and AdoHcy-hydrolase have not been detected in the seminal plasma of man and bull.