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5'-Nucleotidase is involved in sperm capacitation via the cAMP-adenosine pathway and in sperm motility via direct adenosine production from AMP. Since these functions are reduced in varicocele, the aim of this study was to investigate whether the enzyme levels were altered in sperm from varicocele patients. The mean (SD) international units (IU) of 5'-nucleotidase activity in seminal plasma from 35 varicocele III patients was 0.16(0.09) IU ml(-1) vs 0.35(0.13) IU ml(-1) in 53 controls, this decrease being statistically significant at p < or = 0.001. A significant decrease in activity, expressed as international units per mg of protein concentration in spermatozoa homogenates, was also observed with spermatozoa: 0.0018(0.0017) IU mg(-1) in varicocele III vs 0.0081(0.0060) IU mg(-1) in controls, at p < or = 0.001. Compared to controls, the activity decrease observed both in spermatozoa and seminal plasma from 45 men with varicocele I was not statistically significant at p < or = 0.05. To determine the diagnostic value of 5'-nucleotidase in assessing sperm fertility in varicocele III, we used the likelihood ratios method and best cut-offs were identified in receiver operating characteristic curves. With a prevalence of 36%, the post-test probability of infertility was 91% in spermatozoa and 78% in seminal plasma. The cut-off values of 5'-nucleotidase activity discriminating for fertile/unfertile semen were 0.2 IU ml(-1) in seminal plasma and 0.003 IU mg(-1) of protein in spermatozoa. Overall, determination of 5'-nucleotidase activity, especially in spermatozoa, can be useful to characterize different varicocele degrees as well as the sperm fertility potential.

Ecto-5'-nucleotidase (ecto-5'-NT) is a glycosylphosphatidylinositol-anchored membrane-bound protein that is ubiquitous in mammalian tissues. It is a target for a number of therapeutic drugs since increased levels of the enzyme correlate with various disease states. In this investigation, we describe the properties of a soluble ecto-5'-NT derived from bull seminal plasma. The protein was highly heterogeneous as demonstrated by chromatofocusing and two-dimensional PAGE. Sequencing analyses revealed a truncated polypeptide lacking the glycosylphospatidylinositol attachment site, suggesting that it is produced post-translationally by cleavage at Gln(547) and/or Phe(548). Heterogeneity was largely due to differential glycosylation, especially in the oligosaccharides linked to Asn(403). Significant differences in substrate specificity were observed between isoforms and, on the basis of molecular-modelling studies, were interpreted in terms of variable glycosylation causing steric hindrance of the substrate-binding site. Thus the soluble forms of ecto-5'-NT found in bull seminal plasma are unique both biochemically and structurally, and have a putative role in signalling interactions with spermatozoa following ejaculation and capacitation in the female reproductive tract.

The structure of ecto-5'-nucleotidase from bull seminal plasma, containing a glycosyl-phosphatidylinositol anchor, was studied using mass spectrometry. MALDI-MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for approximately 6000 Da. MALDI-MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N-glycosylation. GC-MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high-mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high-mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl-phosphatidylinositol core contained EtN(P)Man3GlcNH2-myo-inositol(P)-glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylglycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S-S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51-Cys57, Cys353-Cys358, Cys365-Cys387 and Cys476-Cys479. This work resolves details of the structure of ecto-5'-nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl-phosphatidylinositol anchor.

The effects of temperature on the three-dimensional organization and on the secondary structure of GPI-anchored 5'-nucleotidase from bull seminal plasma and of its anchor-less form (solubilized ecto-5'-nucleotidase), obtained after GPI anchor removal by phosphatidylinositol-specific phospholipase C were investigated in parallel by circular dichroism and fluorescence spectroscopy. The structural features of the two enzymes were correlated to their functional properties in the temperature range of 25-90 degrees C. The kinetic data indicated that the enzyme activities were temperature dependent, showing the maximal values at 60 degrees C. The relevant Arrhenius plots were linear in the temperature range of 20-60 degrees C and the activation energies were 44.4 and 51.8 kJ/mol for the solubilized and GPI-anchored 5'-nucleotidase, respectively. The time-course measurements of enzyme activity, in the temperature range of 25-55 degrees C, revealed that the two enzymes were of different thermal stability, the solubilized ectoenzyme showing lower thermal deactivation constants and longer half lives. Fluorescence and near UV circular dichroism spectroscopy showed that temperature increases induced remarkable changes in the protein tertiary structure of the two enzymes, whereas far-UV circular dichroism analysis revealed only a small temperature effect on the protein secondary structure content.

Seminal plasma separated from freshly ejaculated bull semen contains vesicles with a 5'-nucleotidase activity incorporated as an ectoenzyme anchored by glycosyl phosphatidylinositol (GPI). After its extraction from bull seminal plasma vesicles, the protein was purified and reconstituted into hen egg yolk lecithin liposomes obtained through prolonged dialysis of buffered n-octylglucoside detergent solutions of lipid, protein and various effectors against detergent-free solutions. Gel filtration experiments showed that the enzyme incorporated into liposomes in a dimeric form with its two subunits linked by disulfide bridges. In the presence of reduced glutathione, the protein dissociated into monomers and failed to incorporate into liposomes. Electron spin resonance (ESR) experiments, performed with liposomes containing electron spin labels localized at the hydrophilic lipid headgroups (5-doxyl stearic acid) or in the hydrophobic lipid hydrocarbon chains (16-doxyl stearic acid), demonstrated that the incorporation of 5'-nucleotidase resulted in the immobilization of the spin probes. Furthermore, the spectral parameters obtained before and after treatment of 5'-nucleotidase-containing liposomes with phosphatidylinositol-specific phospholipase C (PI-PLC) indicated that the liposome membrane bilayer did not contain protein segments. This supports the well-known ecto-localization of 5'-nucleotidase and rules out a previously reported possibility of a proteic transmembrane anchoring of the enzyme.

A phosphodiesterase from bull seminal plasma was purified to homogeneity. The purification procedure involved sequential column chromatographies on DEAE-Sephadex A-50, ConA-Agarose, chromatofocusing and AMP-Agarose. The final yield was about 20% with a 3000-fold purification. As indicated by chromatofocusing, the enzyme is an acidic protein (pI approximately 4.6) and owing to its interaction with Concanavalin A it is also a glycoprotein. The SDS-PAGE showed that the purified phosphodiesterase seemed to be constituted of a single polypeptide chain of about 125 kDa. The enzyme did not show an absolute substrate specificity. Thus, it was able to hydrolyze 4-nitrophenyl ester of 5'-TMP (but not of 3'-TMP), cAMP, nucleic acids as well as NAD+, ADP and ATP. According to its enzymatic properties, bull seminal plasma phosphodiesterase is to be considered an oligonucleate 5'-nucleotidohydrolase. In addition the seminal plasma phosphodiesterase also showed phosphonate esterase activity.

Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structure of 5'-nucleotidase from bull seminal plasma (BSP). Spectra of protein in both D2O and H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide I band. The protein, which is made up of two apparently identical subunits and which contains two zinc atoms, was studied in its native form, in the presence of dithiotreitol (DTT) and after removal of the two zinc atoms by means of nitrilotriacetic acid (NTA). Deconvolved and second derivative spectra of amide I band showed that the native protein contains mostly beta-sheet structure with a minor content of alpha-helix. The quantitative analysis of the amide I components was performed by a curve-fitting procedure which revealed 54% beta-sheet, 18% alpha-helix, 22% beta-turns and 6% unordered structure. The second derivative and deconvolved spectra of amide I band showed that no remarkable changes in the secondary structure of 5'-nucleotidase were induced by either DTT or NTA. These results were confirmed by the curve-fitting analysis where little or no changes occurred in the relative content of amide I components when the protein was treated with DTT or with NTA. Major changes, however, were observed in the thermal denaturation behavior of the protein. The native protein showed denaturation at temperatures between 70 and 75 degrees C, while the maximum of denaturation was observed between 65 and 70 degrees C and between 55 and 60 degrees C in the presence of NTA and DTT, respectively. The results obtained indicate that the two separate subunits of the protein have essentially the same secondary structure as that of the native enzyme.

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).

In bull seminal plasma 5'-nucleotidase is present in heterogeneous forms. The heterogeneity is abolished by treatment of bull seminal plasma with the detergent sodium cholate. The purified enzyme, which is a glycoprotein, shows an apparent molecular mass of 160 kDa on gel filtration in the presence of 50 mmol sodium cholate and an apparent molecular mass of 72 kDa upon SDS/polyacrylamide-gel electrophoresis. The 5'-nucleotidase of bull seminal plasma is a metalloprotein containing 2 zinc ions per molecule of dimeric protein. The removal of the two zinc ions from the protein results in a completely inactive apoenzyme. The substitution of the endogenous zinc with Co(II) Cu(II) produces a holoenzyme which is slightly activated in the case of Co(II), whereas, in the case of Cu(II) only 65% of the initial activity is recovered. The enzyme has a covalently attached glycosyl-phosphatidylinositol moiety which can be removed by treatment with phosphatidylinositol-specific phospholipase C. ESR studies have indicated a radius of 35 A for the protein and that Cu(II) binds to the metal-free enzyme to a site in which sulphur donors can be excluded.

5'-Nucleotidase from bull seminal plasma is inhibited by dithiothreitol and dithioerythritol. These reactives proved to dissociate the dimeric glycoprotein 5'-nucleotidase of Mr 160 000 into two subunits of apparent Mr 80 000, indicating that the subunits are held together by interchain disulfide bridges. HPLC determinations of cysteic acid and carboxymethylcysteine protein derivatives resulted in 50 +/- 3 half-cystine plus cysteine residues, while 1.9 +/- 0.4 free cysteine residues were estimated by HPLC analysis. The enzyme is inhibited by EDTA and EGTA, and the inhibition appears to be of the non-competitive type for both the chelating agents. Experiments for the enzyme activity recovery by MgCl2 and CaCl2 additions, after the EDTA and EGTA treatments in the presence of 8 M urea, are reported.

5'-Nucleotidase of bull seminal plasma has been spin labeled with the sulfhydryl reagent 3-maleimidoproxyl. ESR analysis reveals the presence of two classes of labeled sites. The first is characterized by a long spin label rotational correlation time, from which a protein diameter of about 70 A can be estimated, under the assumption of a spherical shape. The second class is characterized by a shorter correlation time of the covalently bound spin labels and binding of the substrate sodium thymidine 5'-monophosphate to 5'-nucleotidase results in a reduction of their mobility. Low-temperature ESR analysis shows that no paramagnetic ion is bound to the native protein.

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.

5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) occurs in bull seminal plasma in multiple forms. The heterogeneity does not reflect the existence of true isoenzymes, but is due to the association of the enzyme with particulate material and to molecular aggregation phenomena. Addition of detergents to native bull seminal plasma prevents molecular aggregation, solubilizes the particulate form of the enzyme, and results in the appearance of a single molecular form of the enzyme. Enzyme purification can be achieved after three chromatographic steps which involve negative adsorption of 5'-nucleotidase activity on DEAE-Sephadex A-50 followed by two affinity chromatographies on concanavalin A-Sepharose 4B and ADP-agarose. The enzyme appears to be a dimeric glycoprotein. Some properties of the enzyme, including substrate specificity and the effects of hydrogen ion concentration and of various divalent cations, are reported.