5'-nucleotidase from bull seminal plasma

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.     

ESR study of 5'-nucleotidase from bull seminal plasma

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.     

Mass spectrometry study of ecto-5'-nucleotidase from bull seminal plasma.

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.     

Structural and functional relationships in 5'-nucleotidase from bull seminal plasma. A Fourier transform infrared study.

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 bull seminal plasma, chicken gizzard and snake venom is a zinc metalloprotein

Using flame atomic absorption spectrometry the tight association of zinc to three different purified 5'-nucleotidases at a molar ratio of 2 could be proven. These 5'-nucleotidases purified from bull seminal plasma (BSP), chicken gizzard (CG) and snake venom (SV) are thus zinc metalloproteins. Removal of zinc results in the loss of their AMPase activity, which could be fully restored after readdition of zinc at a molar ratio of 2, for BSP and CG, and 1.5, for SV 5'-nucleotidase. Reactivation of their AMPase activity after the removal of zinc could also be obtained by addition of cobalt and copper ions, which were found to also bind with a molar ratio of 2 to the three 5'-nucleotidases tested.     

Effects of dithiothreitol, dithioerythritol and chelating agents on 5'-nucleotidase from bull seminal plasma

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.     

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.     

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).     

5'-nucleotidase: an ecto-enzyme of frog skeletal muscle.

Using 1-4C-labeled AMP and IMP as substrates, 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity was detected at the external surface of frog skeletal muscle with the active site facing toward the extracellular space. The enzyme was firmly bound to the muscle membrane. Its activity was dependent on Ca2+ or Mg2+ and was inhibited by non-radioactive ribonucleoside 5'-monophosphates, or theophylline, while adenosine 3'-monophosphate and p-nitrophenylphosphate had little or no effect. 5'-Nucleotidase with similar properties was also found in the isolated plasma membrane fraction of the muscle.     

Concanavalin A inhibition of ecto-5'-nucleotidase of intact cultured C6 glioma cells.

A pronounced effect of concanavalin A (Con A) upon activity of ecto-5'-nucleotidase of intact C6 glioma cells in culture has been demonstrated. A near linear rate of decrease in 5'-nucleotidase activity was observed upon treatment with concentrations of Con A up to 0.25 muM. Nonspecific phosphatase activity and Ca2+-dependent ATPase activity were not inhibited by Con A treatment of the cells. Of the total 5'-nucleotidase activity of C6 cells (Vmax = 5.0 mumol of Pi liberated/mg of cell protein/hour), approximately 20% still remained after treatment with high concentrations of Con A. The inhibitory effect of Con A operated to reduce substantially Vmax for ecto-5'-nucleotidase. Inhibition was reversed by briefly incubating the Con A-treated cells with alpha-methyl-D-glucoside, or alpha-methyl-D-mannoside, the later being more effective. These findings suggest that a relatively specific, reversible, inhibition of ecto-5'-nucleotidase results from Con A binding to the surface of the intact cultured mammalian cells.     

Purification and partial characterization of the soluble low Km 5'-nucleotidase from human seminal plasma

Soluble low Km 5'-nucleotidase from human seminal plasma has been purified to homogeneity by one affinity and two gel-filtration chromatographic steps. The pure enzyme had a specific activity of 2000 nmol min-1 mg-1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified low Km 5'-nucleotidase revealed a single polypeptide band of 40 +/- 7 kDa and a tetrameric structure of 160 +/- 10 kDa has been proposed for the native enzyme. The kinetic properties of low Km 5'-nucleotidase have been determined and rather unique characteristics have been found for this soluble low Km 5'-nucleotidase: the substrate efficiency was slightly higher for IMP with an optimum pH at 7.5; the enzyme showed an absolute dependence on Mg2+ ions. Ca2+ could replace Mg2+ ions for activity while other divalent cations could not substitute for Mg2+; the enzymes were equally activated by ATP and ADP up to 0.1 mM concentrations. At higher concentrations up to 1 mM, ADP was still an activator while ATP caused a gradual decrease of activation to the native activity. This effect could not be related to the Mg-ATP = complexes since the enzymic preparation Mg(2+)-free still showed the same biphasic pattern of activation.     

Isolation and properties of a phosphatidylcholine-specific phospholipase C from bull seminal plasma

A phospholipase C which hydrolyzes [14C]phosphatidylcholine has been purified 1782-fold from 70% ammonium sulfate extract of bull seminal plasma. Purification steps included acid precipitation, chromatography on DEAE-Sephacel, concanavalin A, octyl-Sepharose 4B and Ultrogel AcA 34. The final step provided homogeneous phospholipase C as determined by polyacrylamide gel electrophoresis. The enzyme comprised two subunits, Mr 69,000 and Mr 55,000, respectively. The enzyme had an optimum at pH 7.2 and pI 5.0. EDTA, Cd2+, Pb2+, Ni2+, Fe2+, and Zn2+ inhibited phospholipase C activity. Km and Vmax on p-nitrophenyl phosphorylcholine and phosphatidylcholine substrates were 20 mM and 17 mumol/min/mg of the purified enzyme and 100 microM and 18 mumol/min/mg of the purified enzyme, respectively. The enzyme appeared to be localized in the acrosome as judged by the binding of anti-phospholipase C to the acrosome. This phospholipase C, unlike other known phospholipases (C), did not hydrolyze [1-14C]phosphatidylinositol. The testicular extract of the guinea pig contained inactive phospholipase C which was activated on incubation with acrosin and trypsin but not chymotrypsin.     

Alpha-L-fucosidase from bull seminal plasma: its purification and acrosome reaction promoting property

Alpha-L-fucosidase was purified from the bull seminal plasma by chromatography on DEAE-disk, octyl sepharose hydrophobic column and HPLC. The enzyme appeared to be pure as judged by the polyacrylamide gel electrophoresis both under the nondenaturing and denaturing conditions. The pure enzyme promoted the acrosome reaction of guinea pig spermatozoa in vitro. This is the first report showing that an acrosomal enzyme induces acrosome reaction which is an essential pre-requisite for the gamete interaction and fertilization.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy

Background

Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy.

Methods

Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors.

Results

Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors.

Conclusions

In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.     

A comparative study on tissue distribution and metabolic adaptation of IMP-GMP 5'-nucleotidase.

1. Activity of a cytoplasmic 5'-nucleotidase which preferentially hydrolyzes IMP and GMP (IMP-GMP 5'-nucleotidase) was determined by a specific immunochemical method in two species of birds and two species of mammals. 2. The activity was markedly high in avian liver, and it increased two-fold in response to a high protein diet in chicken liver. 3. In mammals, the activity was high in testis and spleen. In the rat, the activities in liver, kidney and heart extracts increased by about 30% in response to the high protein diet, while they increased three-fold in regenerating liver. 4. Low activities were detected in skeletal muscles and in erythrocytes of all the species studied.