Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
Effects of dithiothreitol, dithioerythritol and chelating agents on 5′-nucleotidase from bull seminal plasma
Abstract
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.
References (16)
- H. Fukui et al.
- C. Fini et al.
Biochim. Biophys. Acta
(1983) - M.B. Sorensen et al.
Arch. Biochem. Biophys.
(1983) - Y. Worku et al.
FEBS Lett.
(1984) - P.L. Ipata
Anal. Biochem.
(1967) - S. Moore
J. Biol. Chem.
(1963) - A.M. Crestfield et al.
J. Biol. Chem.
(1963) - H. Umagat et al.
J. Chromatogr.
(1982)
Cited by (13)
The ecto-5'-nucleotidase subunits in dimers are not linked by disulfide bridges but by non-covalent bonds
2000, Biochimica et Biophysica Acta - Protein Structure and Molecular EnzymologyIt has long been considered that ecto-5′-nucleotidase (eNT) dimers consist of subunits linked by disulfide bonds. Hydrophilic (6.7S) and amphiphilic (4.0S) dimers were separated by sedimentation analysis of eNT purified from bull seminal plasma. Hydrophilic (4.2S) and amphiphilic (2.6S) eNT monomers were obtained after reduction of disulfide bonds in dimers. The amphiphilic eNT dimers or monomers were converted into their hydrophilic variants with phosphatidylinositol-specific phospholipase C. SDS–PAGE plus Western blot showed 68 kDa subunits, regardless of the addition of β-mercaptoethanol to the SDS mixture. Active eNT monomers were obtained by addition of 1 M guanidinium chloride (Gdn) to dimers, and unfolded subunits by addition of 4 M Gdn. The results unambiguously demonstrate that the subunits in eNT dimers are not linked by disulfide bridges, but by non-covalent bonds, and that dissociation precedes inactivation and unfolding.
Biochemical properties of 5'-nucleotidase from mouse skeletal muscle
1998, Biochimica et Biophysica Acta - Protein Structure and Molecular EnzymologyEcto-5′-nucleotidase (eNT) from mouse muscle has been purified after extraction with detergent followed by chromatography on concanavalin A- and AMP-Sepharose. Three fractions were recovered: UF was NT non-retained in immobilised AMP; F-I was bound enzyme eluted with β-glycerophosphate, and F-II was bound NT released with AMP. eNT was 80 000-fold purified in F-II, this fraction showing proteins of 74, 68 and 51 kDa after immunoblotting. NT in UF migrated at 6.7S after centrifugation in sucrose gradients with Triton X-100, the peak being split into two of 6.7S and 4.4S in gradients with Brij 96. Ecto-NT in F-I or F-II migrated at 5.8S in Triton X-100-, or 4.4S in Brij 96-containing gradients. The hydrodynamic behaviour, concentration in Triton X-114, binding to phenyl-agarose, and sensitivity to phosphatidylinositol-specific phospholipase C revealed that enzyme forms in F-I or F-II were amphiphilic dimers with linked phosphatidylinositol residues, whilst most of NT forms in UF were hydrophilic dimers. A zinc/protein molar ratio of 2.2 was determined for eNT in F-II. NT activity was decreased in assays made in imidazole buffer, and was partly restored with 10 μM Zn2+ or 100 μM Mn2+. In assays with Tris buffer, NT showed a Km for AMP of 12 μM, and was competitively inhibited by ATP or ADP.
Inhibition of ecto-5′-nucleotidase by nitric oxide donors: Implications in renal epithelial cells
1996, Journal of Biological ChemistryWe evaluated, in renal epithelial cells with a proximal tubule phenotype, the effect of nitric oxide (NO) on ecto-5′-nucleotidase (5′-NU), the underlying mechanism and its functional consequence. Sodium nitroprusside (SNP, 1-1000 μM), a NO donor, inhibited 5′-NU activity in a time- and concentration-dependent manner. Consequently, NO blunted the inhibition by extracellular cyclic AMP (cAMP, 10-1000 μM) of sodium-phosphate cotransport, a pathway which involves degradation of adenosine monophosphate (AMP) by 5′-NU. SNP-induced inhibition of 5′-NU was not mediated by cyclic GMP, since it was not mimicked by atrial natriuretic peptide, and was reproduced by isosorbide dinitrate and sodium nitrate, two NO donors. SNP and genuine NO decreased the activity of 5′-NU in renal homogenates, and the effect of SNP was potentiated by dithiothreitol and glutathione, but not by nicotinamide adenine dinucleotide. In vivo in rats, kidney ischemia/reperfusion, which activates inducible NO-synthase, inhibited renal 5′-NU. This inhibition was prevented by Nω-nitro-L-arginine methyl ester, a NO-synthase inhibitor. These results indicate that: (i) NO-related activity inhibited the activity of an ecto-enzyme, 5′-NU, most likely through S-nitrosylation of the enzyme; (ii) inhibition of 5′-NU activity by NOx, which can occur in vivo under pathophysiological conditions, affected the extent to which extracellular cAMP inhibited sodium-Pi cotransport.
Limited proteolysis of chicken gizzard 5′-nucleotidase
1992, Biochimica et Biophysica Acta (BBA)/Protein Structure and MolecularChicken gizzard 5′-nucleotidase represents an ectoenzyme which is linked to the plasma membrane via a phosphatidylinositol glycan. We have characterized the possible domain-like organization of 5′-nucleotidase by limited proteolysis. A hydrophobic proteolytic fregment carrying the intact C-terminus, as well as two major hydrophilic productsm were identified. We developed procedures for specific radiolabelling of the active center of 5′-nucleotidase. This allowed us to locate the catalytic site within hydrophilic fragments obtained after limited proteolysis. We demonstrate that removal of N-linked carbohydrate chains increases the sensitivity of 5′-nucleotidase to proteolytic attack, indicating that sugar moieties protect against proteolysis. 5′-nucleotidase the sensitivity of 5′-nucleotidase to proteolytic attack, indicating that sugar moieties protect against proteolysis. 5′-Nucleotidase represents a binding protein for component of the extracellular matrix. The interaction between 5′-nucleotidase and the laminin/nidogen complex unmasked proteolytic cleavage sites in the C-terminal portion of the enzyme. This resulted in the specific production of a hydrophilic form of 5′-nucleotidase. In summary, we have further charcterized chicken gizzard 5′-nucleotidase: (1) the protein is organized into two domain-like structures, (2) the N-terminal domain harbors the active center; (3) N-linked carbohydrates protect the protein against proteolytic degradation; (4) interaction with components of the extracellular matrix alters the conformation of 5′-nucleotidase.
Structural and functional relationships in 5′-nucleotidase from bull seminal plasma. A Fourier transform infrared study
1992, Biochimica et Biophysica Acta (BBA)/Protein Structure and MolecularFourier 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 β-sheet structure with a minor content of α-helix. The quantitative analysis of the amide I components was performed by a curve-fitting procedure which revealed 54% β-sheet, 18% α-helix, 22% β-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°C, while the maximum of denaturation was observed between 65 and 70°C and between 55 and 60°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.
Purification of 5′-nucleotidase from human seminal plasma
1991, BBA - General Subjects5′-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).