The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
Antonio Raggi - One of the best experts on this subject based on the ideXlab platform.
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role of the hprg component of striated muscle AMP Deaminase in the stability and cellular behaviour of the enzyme
Biomolecules, 2018Co-Authors: Francesca Ronca, Antonio RaggiAbstract:Multiple muscle-specific isoforms of the Zn2+ metalloenzyme AMP Deaminase (AMPD) have been identified based on their biochemical and genetic differences. Our previous observations suggested that the metal binding protein histidine-proline-rich glycoprotein (HPRG) participates in the assembly and maintenance of skeletal muscle AMP Deaminase (AMPD1) by acting as a zinc chaperone. The evidence of a role of millimolar-strength phosphate in stabilizing the AMPD-HPRG complex of both AMPD1 and cardiac AMP Deaminase (AMPD3) is suggestive of a physiological mutual dependence between the two subunit components with regard to the stability of the two isoforms of striated muscle AMPD. The observed influence of the HPRG content on the catalytic behavior of the two enzymes further strengthens this hypothesis. Based on the preferential localization of HPRG at the sarcomeric I-band and on the presence of a Zn2+ binding motif in the N-terminal regions of fast TnT and of the AMPD1 catalytic subunit, we advance the hypothesis that the Zn binding properties of HPRG could promote the association of AMPD1 to the thin filament.
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Role of troponin T and AMP Deaminase in the modulation of skeletal muscle contraction
Rendiconti Lincei, 2017Co-Authors: Francesca Ronca, Antonio RaggiAbstract:In fast muscle, isoforms of troponin T (TnT) contain an N-terminal hypervariable region that does not bind any protein of the thin filament. The N-terminal domain of TnT is removed by calpain during stress conditions and so could modulate the role of TnT in the regulation of contraction by affecting the TnT-binding affinity for tropomyosin (Tm) depending on the sequence and charge within the domain. During skeletal muscle contraction, the myokinase reaction is displaced by AMP Deaminase (AMPD), an allosteric metalloenzyme, toward the formation of ATP. An unrestrained AMPD activity follows the proteolytic cleavage of the enzyme in vivo that releases a 97 aa N-terminal fragment, removing the inhibition exerted by the binding of ATP to a zinc site in the N-terminal region. Rabbit fast TnT or its phosphorylated 50-aa residue N-terminal peptide restores in AMPD the inhibition by ATP, removed in vitro by the release of a 95 aa N-terminal fragment by trypsin. Since the N-terminal region of fast rabbit TnT contains a putative zinc-binding motif, it can be inferred that TnT mimics the regulatory action exerted in native AMPD by the N-terminal domain that holds the enzyme in a less active conformation due to the presence of a zinc ion connecting the N-terminal and C-terminal regions. Together with evidence that AMPD is localized on the myofibril, the data reported in this review on the interactions between AMPD and TnT strongly suggest that these proteins mutually combine to fine-tune the regulation of muscle contraction in fast muscle.
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the role of histidine proline rich glycoprotein as zinc chaperone for skeletal muscle AMP Deaminase
Biomolecules, 2014Co-Authors: Maria Ranieriraggi, Arthur J G Moir, Antonio RaggiAbstract:Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP Deaminase (AMPD) and the metal binding protein histidine-proline-rich glycoprotein (HPRG) acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS) performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated.
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characterization of the metallocenter of rabbit skeletal muscle AMP Deaminase evidence for a dinuclear zinc site
Biochimica et Biophysica Acta, 2007Co-Authors: Stefano Mangani, Maria Ranieriraggi, A Sabbatini, Arthur J G Moir, Daniela Martini, Manuela Benvenuti, Antonio RaggiAbstract:XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP Deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD.
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a calpain like proteolytic activity produces the limited cleavage at the n terminal regulatory domain of rabbit skeletal muscle AMP Deaminase evidence of a protective molecular mechanism
Biochimica et Biophysica Acta, 2004Co-Authors: Daniela Martini, Maria Ranieriraggi, U Montali, A Sabbatini, Arthur J G Moir, S J Thorpe, Antonio RaggiAbstract:On storage at 4 degrees C, rabbit skeletal muscle AMP Deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP Deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains. In addition, the cleavage of a specific fluorogenic peptide substrate by rabbit m-calpain is inhibited by a synthetic peptide that corresponds to residues 10-17 of rabbit skeletal muscle AMP Deaminase; this peptide contains a sequence (K-E-L-D-D-A) that is present in the fourth subdomain A of rabbit calpastatin, suggesting that the N-terminus of AMP Deaminase shares with calpastatin a regulatory sequence that might exert a protective role against the fragmentation-induced activation of AMP Deaminase. These observations suggest that a calpain-like proteinase present in muscle removes from AMP Deaminase a domain that holds the enzyme in an inactive conformation and which also contains a regulatory region that protects against unregulated proteolysis. We conclude that proteolysis of AMP Deaminase is the basis of the large ammonia accumulation that occurs in skeletal muscle subjected to strong tetanic contraction or passing into rigor mortis.
Maria Ranieriraggi - One of the best experts on this subject based on the ideXlab platform.
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the role of histidine proline rich glycoprotein as zinc chaperone for skeletal muscle AMP Deaminase
Biomolecules, 2014Co-Authors: Maria Ranieriraggi, Arthur J G Moir, Antonio RaggiAbstract:Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP Deaminase (AMPD) and the metal binding protein histidine-proline-rich glycoprotein (HPRG) acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS) performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated.
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characterization of the metallocenter of rabbit skeletal muscle AMP Deaminase evidence for a dinuclear zinc site
Biochimica et Biophysica Acta, 2007Co-Authors: Stefano Mangani, Maria Ranieriraggi, A Sabbatini, Arthur J G Moir, Daniela Martini, Manuela Benvenuti, Antonio RaggiAbstract:XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP Deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD.
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immunohistochemical analysis of human skeletal muscle AMP Deaminase deficiency evidence of a correlation between the muscle hprg content and the level of the residual AMP Deaminase activity
Journal of Muscle Research and Cell Motility, 2006Co-Authors: A Sabbatini, Maria Ranieriraggi, Arthur J G Moir, Daniela Martini, Antonio Toscano, M Aguennouz, Enza Polizzi, Alba Migliorato, Olimpia Musumeci, Giuseppe VitaAbstract:We have previously described that, in healthy human skeletal muscle, an anti-histidine-proline-rich-glycoprotein (HPRG) antibody selectively binds to type IIB fibers that are well known to contain the highest level of AMP Deaminase (AMPD) activity, suggesting an association of the HPRG-like protein to the enzyme isoform M. The present paper reports an immunohistochemical study performed on human skeletal muscle biopsies from patients with AMPD deficiency and carried out utilizing both the anti-HPRG antibody and an anti-AMPD antibody specific for the isoform M. A correlation between the muscle content of the HPRG-like protein and the level of AMPD activity was demonstrated. In the specimens from patients with Acquired AMPD deficiency the HPRG-immunoreactivity was less intense than that shown by the control subjects and was related to the residual AMPD activity. The patients affected by Primary and Coincidental AMPD deficiency, which were characterized by an absence of enzyme activity and AMPD immunoreactivity, showed the lowest HPRG immunoreactivity that was clearly detectable by Western blot analysis, but not by immunohistochemistry. The interpretation of the significance of these observations suggests a physiological mutual dependence between skeletal muscle HPRG and AMPD polypeptides with regard to their stability.
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a calpain like proteolytic activity produces the limited cleavage at the n terminal regulatory domain of rabbit skeletal muscle AMP Deaminase evidence of a protective molecular mechanism
Biochimica et Biophysica Acta, 2004Co-Authors: Daniela Martini, Maria Ranieriraggi, U Montali, A Sabbatini, Arthur J G Moir, S J Thorpe, Antonio RaggiAbstract:On storage at 4 degrees C, rabbit skeletal muscle AMP Deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP Deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains. In addition, the cleavage of a specific fluorogenic peptide substrate by rabbit m-calpain is inhibited by a synthetic peptide that corresponds to residues 10-17 of rabbit skeletal muscle AMP Deaminase; this peptide contains a sequence (K-E-L-D-D-A) that is present in the fourth subdomain A of rabbit calpastatin, suggesting that the N-terminus of AMP Deaminase shares with calpastatin a regulatory sequence that might exert a protective role against the fragmentation-induced activation of AMP Deaminase. These observations suggest that a calpain-like proteinase present in muscle removes from AMP Deaminase a domain that holds the enzyme in an inactive conformation and which also contains a regulatory region that protects against unregulated proteolysis. We conclude that proteolysis of AMP Deaminase is the basis of the large ammonia accumulation that occurs in skeletal muscle subjected to strong tetanic contraction or passing into rigor mortis.
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characterization of the zinc binding site of the histidine proline rich glycoprotein associated with rabbit skeletal muscle AMP Deaminase
Journal of Biological Chemistry, 2003Co-Authors: Stefano Mangani, Maria Ranieriraggi, Arthur J G Moir, Daniela Martini, Wolfram Meyerklaucke, Antonio RaggiAbstract:The AMP Deaminase-associated variant of histidine-proline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP Deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zinc-binding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn2+ ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine. 11 cysteines of HPRG from different species are totally conserved, suggesting that five disulfide bridges are essential for the proper folding of the protein. At least another cysteine is present at different positions in the histidine-proline-rich domain of HPRG in all species, suggesting that this cysteine is the candidate for zinc ligation in the muscle variant of HPRG. The same conclusion is likely to be true for the six histidines used by the protein as zinc ligands. The presence in muscle HPRG of a specific zinc-binding site permits us to envisage the addition of HPRG into the family of metallochaperones. In this view, HPRG may enhance the in vivo stability of metalloenzymes such as AMP Deaminase.
Richard L Sabina - One of the best experts on this subject based on the ideXlab platform.
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synthesis and biochemical testing of 3 carboxyphenylethyl imidazo 2 1 f 1 2 4 triazines as inhibitors of AMP Deaminase
ACS Medicinal Chemistry Letters, 2010Co-Authors: Stephen D. Lindell, Simon Maechling, Richard L SabinaAbstract:C-Ribosyl imidazo[2,1-f][1,2,4]triazines and 3-[2-(3-carboxyphenyl)ethyl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ols represent two classes of known AMP Deaminase inhibitors. A combination of the aglycone from the former class with the ribose phosphate mimic from the latter led to the 3-[2-(3-carboxyphenyl)ethyl]imidazo[2,1-f][1,2,4]triazines, which represent a new class of AMP Deaminase inhibitors. The best compound, 3-[2-(3-carboxy-5,6,7,8-tetrahydronaphthyl)ethyl]imidazo[2,1-f][1,2,4]triazine (8), was a good inhibitor of all three human AMPD recombinant isozymes (AMPD1, AMPD2, and AMPD3; IC50 = 0.9−5.7 μM) but a poor inhibitor of the plant recombinant enzyme (Arabidopsis FAC1; IC50 = 200 μM).
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AMP Deaminase deficiency is associated with lower sprint cycling performance in healthy subjects
Journal of Applied Physiology, 2007Co-Authors: Helene Fischer, Richard L Sabina, Mona Esbjornsson, Anna Stromberg, Myriam Peyrardjanvid, Barbara NormanAbstract:AMP Deaminase (AMPD) deficiency is an inherited disorder of skeletal muscle found in ∼2% of the Caucasian population. Although most AMPD-deficient individuals are asymptomatic, a small subset has e...
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calcium activates erythrocyte AMP Deaminase isoform e AMPd3 through a protein protein interaction between calmodulin and the n terminal domain of the AMPd3 polypeptide
Biochemistry, 2005Co-Authors: Donna K Mahnke, Richard L SabinaAbstract:Erythrocyte AMP Deaminase [isoform E (AMPD3)] is activated in response to increased intracellular calcium levels in Tarui's disease, following exposure of ionophore-treated cells to extracellular calcium, and by the addition of calcium to freshly prepared hemolysates. However, the assumption that Ca2+ is a positive effector of isoform E is inconsistent with the loss of sensitivity to this divalent cation following dilution of erythrocyte lysates or enzyme purification. Ca2+ regulation of isoform E was studied by examining in vitro effects of calmodulin (CaM) on this enzyme and by monitoring the influence of CaM antagonists on purine catabolic flow in freshly prepared erythrocytes under various conditions of energy imbalance. Erythrocyte and recombinant isoform E both adsorb to immobilized Ca2+-CaM, and relative adsorption across a series of N-truncated recombinant enzymes localizes CaM binding determinants to within residues 65−89 of the AMPD3 polypeptide. Ca2+-CaM directly stimulates isoform E catalytic ...
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localization of n terminal sequences in human AMP Deaminase isoforms that influence contractile protein binding
Biochemical and Biophysical Research Communications, 2001Co-Authors: Donna K Mahnkezizelman, Richard L SabinaAbstract:Abstract The reversible association of AMP Deaminase (AMPD, EC 3.5.4.6) with elements of the contractile apparatus is an identified mechanism of enzyme regulation in mammalian skeletal muscle. All three members of the human AMPD multigene family contain coding information for polypeptides with divergent N-terminal and conserved C-terminal domains. In this study, serial N-terminal deletion mutants of up to 111 (AMPD1), 214 (AMPD2), and 126 (AMPD3) residues have been constructed without significant alteration of catalytic function or protein solubility. The entire sets of active enzymes are used to extend our understanding of the contractile protein binding of AMPD. Analysis of the most truncated active enzymes demonstrates that all three isoforms can associate with skeletal muscle actomyosin and suggests that a primary binding domain is located within the C-terminal 635–640 residues of each polypeptide. However, discrete stretches of N-terminal sequence alter this behavior. Residues 54–83 in the AMPD1 polypeptide contribute to a high actomyosin binding capacity of both isoform M spliceoforms, although the exon 2− enzyme exhibits significantly greater association compared to its exon 2+ counterpart. Conversely, residues 129–183 in the AMPD2 polypeptide reduce actomyosin binding of isoform L. In addition, residues 1–48 in the AMPD3 polypeptide dramatically suppress contractile protein binding of isoform E, thus allowing this enzyme to participate in other intracellular interactions.
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towards an understanding of the functional significance of n terminal domain divergence in human AMP Deaminase isoforms
Pharmacology & Therapeutics, 2000Co-Authors: Richard L Sabina, Donna K MahnkezizelmanAbstract:Human AMP Deaminase (AMPD; EC 3.5.4.6) isoforms are encoded by a multigene family and have conserved C-terminal domains that contain catalytic residues and an ATP-binding site. N-terminal domains diverge dramatically, yet are conserved when compared across mammalian species. Cross-species conservation of entire gene-specific polypeptides (e.g., rat versus human AMPD1) suggests that divergent N-terminal domains may play a role in isoform-specific properties of the enzyme. It now has become evident that the majority of published data used to characterize purified AMPD isoforms were likely derived from preparations lacking significant portions of their N-terminal domains (up to nearly 100 residues). Accumulating evidence indicates that divergent N-terminal sequences do influence catalytic behavior, protein-protein interactions, and intracellular distributions of this enzyme.
Arthur J G Moir - One of the best experts on this subject based on the ideXlab platform.
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the role of histidine proline rich glycoprotein as zinc chaperone for skeletal muscle AMP Deaminase
Biomolecules, 2014Co-Authors: Maria Ranieriraggi, Arthur J G Moir, Antonio RaggiAbstract:Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP Deaminase (AMPD) and the metal binding protein histidine-proline-rich glycoprotein (HPRG) acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS) performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated.
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characterization of the metallocenter of rabbit skeletal muscle AMP Deaminase evidence for a dinuclear zinc site
Biochimica et Biophysica Acta, 2007Co-Authors: Stefano Mangani, Maria Ranieriraggi, A Sabbatini, Arthur J G Moir, Daniela Martini, Manuela Benvenuti, Antonio RaggiAbstract:XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP Deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD.
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immunohistochemical analysis of human skeletal muscle AMP Deaminase deficiency evidence of a correlation between the muscle hprg content and the level of the residual AMP Deaminase activity
Journal of Muscle Research and Cell Motility, 2006Co-Authors: A Sabbatini, Maria Ranieriraggi, Arthur J G Moir, Daniela Martini, Antonio Toscano, M Aguennouz, Enza Polizzi, Alba Migliorato, Olimpia Musumeci, Giuseppe VitaAbstract:We have previously described that, in healthy human skeletal muscle, an anti-histidine-proline-rich-glycoprotein (HPRG) antibody selectively binds to type IIB fibers that are well known to contain the highest level of AMP Deaminase (AMPD) activity, suggesting an association of the HPRG-like protein to the enzyme isoform M. The present paper reports an immunohistochemical study performed on human skeletal muscle biopsies from patients with AMPD deficiency and carried out utilizing both the anti-HPRG antibody and an anti-AMPD antibody specific for the isoform M. A correlation between the muscle content of the HPRG-like protein and the level of AMPD activity was demonstrated. In the specimens from patients with Acquired AMPD deficiency the HPRG-immunoreactivity was less intense than that shown by the control subjects and was related to the residual AMPD activity. The patients affected by Primary and Coincidental AMPD deficiency, which were characterized by an absence of enzyme activity and AMPD immunoreactivity, showed the lowest HPRG immunoreactivity that was clearly detectable by Western blot analysis, but not by immunohistochemistry. The interpretation of the significance of these observations suggests a physiological mutual dependence between skeletal muscle HPRG and AMPD polypeptides with regard to their stability.
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a calpain like proteolytic activity produces the limited cleavage at the n terminal regulatory domain of rabbit skeletal muscle AMP Deaminase evidence of a protective molecular mechanism
Biochimica et Biophysica Acta, 2004Co-Authors: Daniela Martini, Maria Ranieriraggi, U Montali, A Sabbatini, Arthur J G Moir, S J Thorpe, Antonio RaggiAbstract:On storage at 4 degrees C, rabbit skeletal muscle AMP Deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP Deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains. In addition, the cleavage of a specific fluorogenic peptide substrate by rabbit m-calpain is inhibited by a synthetic peptide that corresponds to residues 10-17 of rabbit skeletal muscle AMP Deaminase; this peptide contains a sequence (K-E-L-D-D-A) that is present in the fourth subdomain A of rabbit calpastatin, suggesting that the N-terminus of AMP Deaminase shares with calpastatin a regulatory sequence that might exert a protective role against the fragmentation-induced activation of AMP Deaminase. These observations suggest that a calpain-like proteinase present in muscle removes from AMP Deaminase a domain that holds the enzyme in an inactive conformation and which also contains a regulatory region that protects against unregulated proteolysis. We conclude that proteolysis of AMP Deaminase is the basis of the large ammonia accumulation that occurs in skeletal muscle subjected to strong tetanic contraction or passing into rigor mortis.
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characterization of the zinc binding site of the histidine proline rich glycoprotein associated with rabbit skeletal muscle AMP Deaminase
Journal of Biological Chemistry, 2003Co-Authors: Stefano Mangani, Maria Ranieriraggi, Arthur J G Moir, Daniela Martini, Wolfram Meyerklaucke, Antonio RaggiAbstract:The AMP Deaminase-associated variant of histidine-proline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP Deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zinc-binding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn2+ ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine. 11 cysteines of HPRG from different species are totally conserved, suggesting that five disulfide bridges are essential for the proper folding of the protein. At least another cysteine is present at different positions in the histidine-proline-rich domain of HPRG in all species, suggesting that this cysteine is the candidate for zinc ligation in the muscle variant of HPRG. The same conclusion is likely to be true for the six histidines used by the protein as zinc ligands. The presence in muscle HPRG of a specific zinc-binding site permits us to envisage the addition of HPRG into the family of metallochaperones. In this view, HPRG may enhance the in vivo stability of metalloenzymes such as AMP Deaminase.
A Sabbatini - One of the best experts on this subject based on the ideXlab platform.
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characterization of the metallocenter of rabbit skeletal muscle AMP Deaminase evidence for a dinuclear zinc site
Biochimica et Biophysica Acta, 2007Co-Authors: Stefano Mangani, Maria Ranieriraggi, A Sabbatini, Arthur J G Moir, Daniela Martini, Manuela Benvenuti, Antonio RaggiAbstract:XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP Deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD.
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immunohistochemical analysis of human skeletal muscle AMP Deaminase deficiency evidence of a correlation between the muscle hprg content and the level of the residual AMP Deaminase activity
Journal of Muscle Research and Cell Motility, 2006Co-Authors: A Sabbatini, Maria Ranieriraggi, Arthur J G Moir, Daniela Martini, Antonio Toscano, M Aguennouz, Enza Polizzi, Alba Migliorato, Olimpia Musumeci, Giuseppe VitaAbstract:We have previously described that, in healthy human skeletal muscle, an anti-histidine-proline-rich-glycoprotein (HPRG) antibody selectively binds to type IIB fibers that are well known to contain the highest level of AMP Deaminase (AMPD) activity, suggesting an association of the HPRG-like protein to the enzyme isoform M. The present paper reports an immunohistochemical study performed on human skeletal muscle biopsies from patients with AMPD deficiency and carried out utilizing both the anti-HPRG antibody and an anti-AMPD antibody specific for the isoform M. A correlation between the muscle content of the HPRG-like protein and the level of AMPD activity was demonstrated. In the specimens from patients with Acquired AMPD deficiency the HPRG-immunoreactivity was less intense than that shown by the control subjects and was related to the residual AMPD activity. The patients affected by Primary and Coincidental AMPD deficiency, which were characterized by an absence of enzyme activity and AMPD immunoreactivity, showed the lowest HPRG immunoreactivity that was clearly detectable by Western blot analysis, but not by immunohistochemistry. The interpretation of the significance of these observations suggests a physiological mutual dependence between skeletal muscle HPRG and AMPD polypeptides with regard to their stability.
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a calpain like proteolytic activity produces the limited cleavage at the n terminal regulatory domain of rabbit skeletal muscle AMP Deaminase evidence of a protective molecular mechanism
Biochimica et Biophysica Acta, 2004Co-Authors: Daniela Martini, Maria Ranieriraggi, U Montali, A Sabbatini, Arthur J G Moir, S J Thorpe, Antonio RaggiAbstract:On storage at 4 degrees C, rabbit skeletal muscle AMP Deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP Deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains. In addition, the cleavage of a specific fluorogenic peptide substrate by rabbit m-calpain is inhibited by a synthetic peptide that corresponds to residues 10-17 of rabbit skeletal muscle AMP Deaminase; this peptide contains a sequence (K-E-L-D-D-A) that is present in the fourth subdomain A of rabbit calpastatin, suggesting that the N-terminus of AMP Deaminase shares with calpastatin a regulatory sequence that might exert a protective role against the fragmentation-induced activation of AMP Deaminase. These observations suggest that a calpain-like proteinase present in muscle removes from AMP Deaminase a domain that holds the enzyme in an inactive conformation and which also contains a regulatory region that protects against unregulated proteolysis. We conclude that proteolysis of AMP Deaminase is the basis of the large ammonia accumulation that occurs in skeletal muscle subjected to strong tetanic contraction or passing into rigor mortis.
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isolation by zinc affinity chromatography of the histidine proline rich glycoprotein molecule associated with rabbit skeletal muscle AMP Deaminase evidence that the formation of a protein protein complex between the catalytic subunit and the novel co
Biochimica et Biophysica Acta, 2003Co-Authors: Maria Ranieriraggi, A Sabbatini, Arthur J G Moir, Daniela Martini, Antonio RaggiAbstract:The histidine-proline-rich glycoprotein (HPRG) component of rabbit skeletal muscle AMP Deaminase under denaturing and reducing conditions specifically binds to a Zn(2+)-charged affinity column and is only eluted with an EDTA-containing buffer that strips Zn(2+) from the gel. The isolated protein is homogeneous showing an apparent molecular weight (MW) of 95000 and the N-terminal sequence L-T-P-T-D-X-K-T-T-K-P-L-A-E-K-A-L-D-L-I, corresponding to that of rabbit plasma HPRG. The incubation with peptide-N-glycosidase F promotes the reduction of the apparent MW of isolated HPRG to 70000, characterizing it as a N-glycosylated protein. The separation from AMP Deaminase of an 85-kDa component with a blocked N terminus is observed when the enzyme is applied to the Zn-charged column under nondenaturing conditions. On storage under reducing conditions, this component undergoes an 85- to 95-kDa transition yielding a L-T-P-T-D-X-K-T-T-K-P-L N-terminal sequence, suggesting that the shift in the migration on SDS/PAGE as well as the truncation of the protein at its N terminus are promoted by the reduction of a disulfide bond present in freshly isolated HPRG. The separation of HPRG induces a marked reduction in the solubility of AMP Deaminase, strongly suggesting a role of HPRG in assuring the molecular integrity of the enzyme.
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presence in human skeletal muscle of an AMP Deaminase associated protein that reacts with an antibody to human plasma histidine proline rich glycoprotein
Journal of Histochemistry and Cytochemistry, 1999Co-Authors: A Sabbatini, Maria Ranieriraggi, Arthur J G Moir, L Pollina, P Viacava, John R Ashby, Antonio RaggiAbstract:Histidine-proline-rich glycoprotein (HPRG) is a protein that is synthesized by parenchimal liver cells. The protein has been implicated in a number of plasma-specific processes, including blood coagulation and fibrinolysis. We have recently reported the association of an HPRG-like protein with rabbit skeletal muscle AMP Deaminase (AMPD). The results of the immunological analysis reported here demonstrate that an antibody against human plasma HPRG reacts with an AMPD preparation from human skeletal muscle. To probe the localization of the putative HPRG-like protein in human skeletal muscle, serial sections from frozen biopsy specimens were processed for immunohistochemical and histoenzymatic stains. A selective binding of the anti-HPRG antibody to Type IIB muscle fibers was detected, suggesting a preferential association of the novel protein to the AMPD isoenzyme contained in the fast-twitch glycolytic fibers.