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M Stefani

Publications and source records attributed to M Stefani.

At least 19 recordsLinked to original sources
PubMed1996

C-terminal region contributes to muscle acylphosphatase three-dimensional structure stabilisation.

Ser-Ala and Ser-Ala-Ser-Ala C-terminus elongated (delta+2 and delta+4, respectively) and two C-terminus deleted (delta-2 and delta-3) muscle acylphosphatase mutants were investigated to assess the catalytic and structural roles of the C-terminal region. The kinetic analysis of these mutants shows that the removal of two or three C-terminal residues reduces the catalytic activity to 7% and 4% of the value measured for the wild-type enzyme, respectively; instead, the elongation of the C-terminus does not significantly change the enzyme behaviour. 1H Nuclear magnetic resonance spectroscopy indicates that all mutants display a native-like fold though they appear less stable, particularly delta-2 and delta-3 mutants, as compared to the wild-type enzyme. Such destabilisation of the C-terminal modified mutants is further confirmed by urea inactivation experiments. The results here presented account for an involvement of the C-terminal region in the stabilisation of the three-dimensional structure of acylphosphatase, particularly at the active-site level. Moreover, a participation of the C-terminal carboxyl group to the catalytic mechanism can be excluded.

Acid Anhydride Hydrolases↗
PubMed1995

Properties of N-terminus truncated and C-terminus mutated muscle acylphosphatases.

Enzymatic activity and structure of N-terminus truncated and C-terminus substituted muscle acylphosphatase mutants were investigated by kinetic studies under different conditions and 1H NMR spectroscopy, respectively. The N-terminus truncated mutant lacked the first six residues (delta 6), whereas arginine 97 and tyrosine 98 were replaced by glutamine giving two C-terminus substituted mutants (R97Q and Y98Q, respectively). All acylphosphatase forms were obtained by modifications of a synthetic gene coding for the human muscle enzyme which was expressed in E. coli. The delta 6 deletion mutant elicited a reduced specific activity and a native-like structure. The kinetic and structural properties of R97Q and Y98Q mutants indicate a possible role of Arg-97 in the stabilisation of the active site correct conformation, most likely via back-bone and side chain interactions with Arg-23, the residue involved in phosphate binding by the enzyme. This study also suggests a possible involvement of Tyr-98 in the stabilisation of the acylphosphatase overall structure.

Base Sequence↗
PubMed1994

Equilibrium unfolding studies of horse muscle acylphosphatase.

The stability and equilibrium unfolding behaviour of horse muscle acylphosphatase have been studied by denaturing the protein under various conditions of temperature, pH, and urea concentration. Far-ultraviolet circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy indicate that this small monomeric protein unfolds reversibly and cooperatively. Thermodynamic parameters, the Gibbs free energy delta G and enthalpy delta H of unfolding, have been estimated for denaturation of the protein from NMR and CD data as 19 kJ mol-1 and 350 kJ mol-1, respectively. CD and 1H-NMR results suggest the presence of very little persistent residual structure in the denatured states studied under these different conditions. Furthermore, photo-chemically induced dynamic nuclear polarisation experiments show that in the denatured states aromatic residues are freely accessible to a flavin dye probe.

Acid Anhydride Hydrolases↗
PubMed1994

Arginine-23 is involved in the catalytic site of muscle acylphosphatase.

Three mutants of human muscle acylphosphatase in which arginine-23 was replaced by glutamine, histidine and lysine, respectively, were prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. All mutants, purified by affinity chromatography, were almost completely unable to catalyze the hydrolysis of the substrate. 1H-NMR spectroscopy experiments showed the absence of any major conformational changes of the three mutants with respect to the wild-type recombinant enzyme. Equilibrium dialysis experiments demonstrated that the mutated proteins lost the ability of binding inorganic phosphate, a competitive inhibitor of the enzyme. These results strongly support an involvement of arginine-23 at the phosphate binding-site of acylphosphatase, confirming the hypothesis of the existence of a phosphate binding structural motif recently proposed by other authors.

Acid Anhydride Hydrolases↗
PubMed1994

Aspartic-129 is an essential residue in the catalytic mechanism of the low M(r) phosphotyrosine protein phosphatase.

The crystal structure of the bovine liver low M(r) phosphotyrosine protein phosphatase suggests the involvement of aspartic acid-129 in enzyme catalysis. The Asp-129 to alanine mutant has been prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. The purified mutant elicited an highly reduced specific activity (about 0.04% of the activity of the wild-type) and a native-like fold, as judged by 1H NMR spectroscopy. The kinetic analysis revealed that the mutant is able to bind the substrate and a competitive inhibitor, such as inorganic phosphate. Moreover, trapping experiments demonstrated it maintains the ability to form the E-P covalent complex. The Asp-129 to alanine mutant shows extremely reduced enzyme phosphorylation (k2) and dephosphorylation (k3) kinetic constant values as compared to the wild-type enzyme. The data reported indicate that aspartic acid-129 is likely to be involved both in the first step and in the rate-limiting step of the catalytic mechanism, i.e. the nucleophilic attack of the phosphorylated intermediate.

Animals↗
PubMed1994

The crystal structure of a low-molecular-weight phosphotyrosine protein phosphatase.

Protein tyrosine phosphorylation and dephosphorylation are central reactions for control of cellular division, differentiation and development. Here we describe the crystal structure of a low-molecular-weight phosphotyrosine protein phosphatase (PTPase), a cytosolic phosphatase present in many mammalian cells. The enzyme catalyses the dephosphorylation of phosphotyrosine-containing substrates, and overexpression of the protein in normal and transformed cells inhibits cell proliferation. The structure of the low-molecular-weight PTPase reveals an alpha/beta protein containing a phosphate-binding loop motif at the amino end of helix alpha 1. This motif includes the essential active-site residues Cys 12 and Arg 18 and bears striking similarities to the active-site motif recently described in the structure of human PTP1B. The structure of the low-molecular-weight PTPase supports a reaction mechanism involving the conserved Cys 12 as an attacking nucleophile in an in-line associative mechanism. The structure also suggests a catalytic role for Asp 129 in the reaction cycle.

Animals↗
PubMed1994

Antisense peptides to the 43-57 region of acylphosphatase and to the 46-60 region of two isoenzymes of a low-M(r) phosphotyrosine protein phosphatase do not interact with the corresponding proteins.

Three peptides complementary to exposed regions of two low-M(r) phosphotyrosine protein phosphatase isoenzymes and of the acylphosphatase muscle isoenzyme have been synthesized. Each peptide was synthesized on two different types of resins; the peptides were anchored to the resins by amide linkages. The peptide resins were checked by amino acid analysis and Edman degradation and directly used for enzyme purification. Despite our attempts, none of the resins was able to bind significant amounts of the corresponding protein, indicating the lack of interaction between the three proteins and the corresponding complementary peptides. This result agrees with many other reports, confirming that the molecular-recognition theory has no general validity.

Acid Anhydride Hydrolases↗
PubMed1994

Fetal hydrops due to right ventricular and tricuspid valve dysplasia. A case report.

We report a case of fetal hydrops at 32 weeks of gestation, diagnosed prenatally due to an unusual form of non-Ebstein dysplasia of the tricuspid valve and of the right ventricle, different from the classification used in the literature. Digoxin therapy, administered for two weeks in utero and then after the premature delivery, did not improve the massive right heart failure due to the tricuspid insufficiency, and the baby died at 4 days of life.

Adult↗
PubMed1993

Dephosphorylation of tyrosine phosphorylated synthetic peptides by rat liver phosphotyrosine protein phosphatase isoenzymes.

Five phosphotyrosine-containing peptides have been synthesized by FMOC solid-phase peptide synthesis. These peptides correspond to the 411-419 sequence of the Xenopus src oncogene, to the 1191-1220 sequence of the human EGF receptor precursor, to the 1146-1158 sequence of the human insulin receptor, to the 856-865 sequence of the human beta-PDGF receptor, and to the 5-16 sequence of the erythrocyte human band 3. The peptides were used as substrates for activity assay of two isoforms (AcP1 and AcP2) of a low molecular weight cytosolic PTPase. The assay, performed in microtiter EIA plates using Malachite green to determine the released phosphate, was rapid, reproducible, and sensitive. Both PTPase isoforms were able to hydrolyze all synthesized peptides, though with different affinity and rate. The main kinetic parameters were compared and discussed with respect to the role of the two enzymes in the cell.

Amino Acid Sequence↗
PubMed1992

Expression and turnover of acylphosphatase (muscular isoenzyme) in L6 myoblasts during myogenesis.

Acylphosphatase (muscular isoenzyme) levels have been measured in L6J1 myoblasts either proliferating or differentiating into myotubes. Results indicated that the increase in enzyme levels during differentiation is very similar to that of creatine kinase, a specific muscular enzyme. The half-lives of acylphosphatase in myoblasts and myotubes were also determined; t1/2 values of 3 h 30 min (myoblasts), and 2 h 18 min (myotubes) were found. These results indicate that acylphosphatase could be considered a short-lived muscle-specific protein and that its increase in myotubes must be accompanied by an activation of its breakdown.

Acid Anhydride Hydrolases↗
PubMed1991

Preparation and properties of des-Tyr98 and des-Arg97-Tyr98 acylphosphatase (muscular isoenzyme).

Previous NMR reports indicated that Tyr98, the C-terminal residue of the muscular form of acylphosphatase, is likely to be part of the enzyme's active site. In addition, there is evidence that an arginine residue participates to the catalyzed reaction, possibly as phosphate binding site. Among all Arg residues present in the muscular forms of acylphosphatase, four, i.e. Arg23, Arg74, Arg77, and Arg97, appear to be conserved in all species checked thus far. We prepared the des-Tyr98 and des-Arg97-Tyr98 derivatives of the native acylphosphatase to investigate the properties of both modified enzymes. The enzyme lacking Tyr98 was found to be catalytically less effective than the native one, whereas the des-Arg97-Tyr98 acylphosphatase was completely inactive. This evidence suggests that Arg97 participates directly to the active site catalytic mechanism. Fluorescence and CD spectra revealed that the latter enzyme could have been undergone some conformational change that could account for the loss of activity; on the other hand, the one-dimensional NMR spectra of either native and des-Arg97-Tyr98 enzymes were strictly similar, thus demonstrating that the removal of the two C-terminal residues does not markedly affect the fold of the enzyme. The results reported are proof of a critical contribution of Arg97 to the acylphosphatase active site; however, we cannot exclude that the function of this residue is merely to stabilize the active site conformation and dynamics.

Acid Anhydride Hydrolases↗
PubMed1990

Purification and characterization of acylphosphatase erythrocyte isoenzyme from turkey muscle.

An acylphosphatase has been purified from turkey muscle in a rapid and high-yield way. The enzyme has been characterized for structural, kinetic, and immunological parameters, as well as with regard to its stability to thermal, urea, and phenylglyoxal inactivation. The enzyme is quite different from the turkey muscular isoenzyme, and shows structural and kinetic properties that are very similar to those previously reported for the erythrocyte isoenzyme from human erythrocytes and from chicken muscle. From the data reported it appears that this enzyme corresponds to the acylphosphatase erythrocyte isoenzyme. Unlike the erythrocyte isoenzymes studied so far, this enzyme is able to cross-react with antibodies that are raised against the muscular isoenzyme.

Acid Anhydride Hydrolases↗
PubMed1990

Generation of monoclonal antibodies to human acylphosphatase (muscular isoenzyme) and application in solid-phase immunoassay.

Monoclonal antibodies to human acylphosphatase (muscle isoenzyme) were generated by an improved hybridoma technique. Immunization consisted of four antigen administrations in an overall period of 15 weeks. After cell fusion and repeated subcloning of positive lines, seven monoclonal antibodies with good affinity and specificity were selected. These antibodies were characterized for their affinity constant and immunoreactivity. The latter was determined using peptides generated by CNBr cleavage of the antigen. One of the selected antibodies had an affinity constant such that it could be used to develop a competitive enzyme-linked immunosorbent assay. In our test, the antigen that was coated on the matrix, and the free one, competed for the antibody-horseradish peroxidase conjugate. No cross-reactivity with the erythrocyte iso-enzyme was found, and the test showed a limit in sensitivity of 0.32 ng/ml of antigen. We expect that the enzyme immunoassay could be useful for clinical application.

Acid Anhydride Hydrolases↗
PubMed1990

Immunoaffinity purification and immunoassay determination of human erythrocyte acylphosphatase.

Specific anti-human erythrocyte acylphosphatase antibodies were raised in rabbits, purified by affinity chromatography, and used to develop an enzyme purification procedure based on an immunoaffinity chromatography step. This procedure permitted the rapid purification of the enzyme, with a high final yield and with a specific activity very similar to that found for the enzyme purified by the standard procedure. The noncompetitive enzyme-linked immunoadsorbent assay developed with the affinity-purified antibodies was very specific and sensitive in that a positive reaction could be detected in the presence of antigen amounts of as little as 0.01 ng/ml. By this assay the enzyme content was determined in normal cells, tissues, and organs as well as in blood samples from hemopathy-affected patients. This test could possibly have clinical applications.

Acid Anhydride Hydrolases↗
PubMed1989

Mobility of secondary structure units of horse-muscle acylphosphatase. Relation to antigenicity.

The antigenic properties of acylphosphatase are compared with its various sequential characteristics (hydrophobicity, chemical shift of the main-chain 1H-NMR resonances, numbers and intensities of the nuclear Overhauser enhancements, hydrogen-deuterium exchange and sequential arrangement of the secondary structure units). The discussion is based on the complete sequential assignment of the 1H-NMR spectrum and the knowledge of the three-dimensional fold of the protein obtained by NMR spectroscopy from distance geometry calculations. Regions with very different degrees of mobility can be distinguished. It is found that all major antigenic sites are located in the most mobile surface loops.

Acid Anhydride Hydrolases↗
PubMed1989

The sequence-specific assignment of the 1H-NMR spectrum of an enzyme, horse-muscle acylphosphatase.

A complete range of two-dimensional NMR experiments was used for the assignment of the 1H-NMR spectrum of horse muscle acylphosphatase. Firstly the spin systems of some easily identifiable amino acid side chains were assigned. These side chains involved all the aromatic residues and all the leucine, valine, isoleucine, threonine, alanine, proline as well as some of the glycine residues. Analysis of nuclear Overhauser enhancement spectra in our previous work had identified the sequential and long-range patterns characteristics for secondary structure elements. This result had also provided the identification of the main-chain alpha and amide proton resonances. Several of the completely assigned spin systems were then identified as being part of the secondary structure units which led, after analysis of the primary amino acid sequence, to unambiguous sequence-specific assignments. The identification and assignment of the remaining side-chain resonances was then completed and are reported here. These results provide a complete data base for the three-dimensional structure determination of this enzyme in solution.

Acid Anhydride Hydrolases↗