The Experts below are selected from a list of 156 Experts worldwide ranked by ideXlab platform
Salvatore V. Pizzo - One of the best experts on this subject based on the ideXlab platform.
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Incorporation of non-proteolytic proteins by murine α2-Macroglobulin
Biochimica et biophysica acta, 1999Co-Authors: Gourab Bhattacharjee, Hanne Gron, Salvatore V. PizzoAbstract:Human alpha2-Macroglobulin is a tetrameric glycoprotein with a molecular weight of 718 kDa that is present in human plasma at high concentrations. Murine alpha2-Macroglobulin is homologous to human alpha2-Macroglobulin but it undergoes post-translational cleavage in the subunits. Each subunit of alpha2-Macroglobulin contains a thiolester which can be cleaved by small nucleophiles. In human alpha2-Macroglobulin this results in a conformational change to a receptor-recognized form and a change in the electrophoretic mobility. Recent work has demonstrated that this process is reversible and during this reversal non-proteolytic proteins can become covalently trapped within the human alpha2-Macroglobulin molecule. The present study further investigates this observation and examines the question whether reversal of thiolester cleavage occurs in mouse alpha2-Macroglobulin. Previous studies suggest that small nucleophiles only partially convert mouse alpha2-Macroglobulin to a receptor-recognized form. We demonstrate here that under appropriate conditions, mouse alpha2-Macroglobulin is fully converted by NH3. We also demonstrate that despite structural and kinetic differences between human and mouse alpha2-Macroglobulin, both molecules are able to incorporate non-proteolytic ligands in a similar manner. This leads us to propose a general model of ligand incorporation via nucleophilic exchange in multimeric alpha-Macroglobulins.
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activated human plasma carboxypeptidase b is retained in the blood by binding to alpha2 Macroglobulin and pregnancy zone protein
Journal of Biological Chemistry, 1996Co-Authors: Zuzana Valnickova, Salvatore V. Pizzo, I B Thogersen, Soren Christensen, Jan J. EnghildAbstract:Abstract A 66-kDa glycosylated carboxypeptidase, plasma pro-carboxypeptidase B (pro-plasma CPB), has recently been identified in human blood (Eaton, D. L., Malloy, B. E., Tsai, S. P., Henzel, W., and Drayna, D. (1991) J. Biol. Chem. 266, 21833-21838). The pro-enzyme binds to plasminogen and the active enzyme is specific for COOH-terminal Lys or Arg residues. These properties implicate a role in the fibrinolytic or coagulation system. However, we show that the molecular mass of the active plasma CPB is approximately 36 kDa, which is below the glomerular filtration limit. Since activated plasma CPB no longer binds plasminogen, the active enzyme may not be retained in the circulation. To investigate this, we performed plasma elimination studies in mice which showed that 125I-plasma CPB remains in the circulation despite its small size. Native polyacrylamide gel electrophoresis of blood samples removed from the mice revealed that plasma CPB migrated as a high molecular weight band. Similar bands were observed in vitro when 125I-plasma CPB was added to plasma from humans and other species. The plasma CPB-binding proteins were purified from human plasma and identified as α2-Macroglobulin (α2M) and pregnancy zone protein. Only the active enzyme bound to the two α-Macroglobulins, and the interaction was specific for α2M in its native conformation, but not its receptor recognized forms. The complex between human α2M and plasma CPB dissociated during SDS-polyacrylamide gel electrophoresis and transverse urea gel electrophoresis suggesting that the interaction was noncovalent and depended on the tertiary structure of the native α2M molecule. The catalytic activity of plasma CPB was not significantly affected by its binding to α2M. The specific binding of plasma CPB to α-Macroglobulins suggest that these proteins may function as a “shuttle” in vivo to modulate the clearance of plasma CPB from the circulatory system.
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activated human plasma carboxypeptidase b is retained in the blood by binding to alpha2 Macroglobulin and pregnancy zone protein
Journal of Biological Chemistry, 1996Co-Authors: Zuzana Valnickova, Salvatore V. Pizzo, I B Thogersen, Soren Christensen, Charleen T Chu, Jan J. EnghildAbstract:Abstract A 66-kDa glycosylated carboxypeptidase, plasma pro-carboxypeptidase B (pro-plasma CPB), has recently been identified in human blood (Eaton, D. L., Malloy, B. E., Tsai, S. P., Henzel, W., and Drayna, D. (1991) J. Biol. Chem. 266, 21833-21838). The pro-enzyme binds to plasminogen and the active enzyme is specific for COOH-terminal Lys or Arg residues. These properties implicate a role in the fibrinolytic or coagulation system. However, we show that the molecular mass of the active plasma CPB is approximately 36 kDa, which is below the glomerular filtration limit. Since activated plasma CPB no longer binds plasminogen, the active enzyme may not be retained in the circulation. To investigate this, we performed plasma elimination studies in mice which showed that 125I-plasma CPB remains in the circulation despite its small size. Native polyacrylamide gel electrophoresis of blood samples removed from the mice revealed that plasma CPB migrated as a high molecular weight band. Similar bands were observed in vitro when 125I-plasma CPB was added to plasma from humans and other species. The plasma CPB-binding proteins were purified from human plasma and identified as α2-Macroglobulin (α2M) and pregnancy zone protein. Only the active enzyme bound to the two α-Macroglobulins, and the interaction was specific for α2M in its native conformation, but not its receptor recognized forms. The complex between human α2M and plasma CPB dissociated during SDS-polyacrylamide gel electrophoresis and transverse urea gel electrophoresis suggesting that the interaction was noncovalent and depended on the tertiary structure of the native α2M molecule. The catalytic activity of plasma CPB was not significantly affected by its binding to α2M. The specific binding of plasma CPB to α-Macroglobulins suggest that these proteins may function as a “shuttle” in vivo to modulate the clearance of plasma CPB from the circulatory system.
Poul Erik Hyldgaard Jensen - One of the best experts on this subject based on the ideXlab platform.
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Aberrant forms of α2-Macroglobulin purified from patients with multiple sclerosis
Clinica Chimica Acta, 2000Co-Authors: Martin Gunnarsson, Torgny Stigbrand, Poul Erik Hyldgaard JensenAbstract:Abstract The biochemical properties of α2-Macroglobulin were investigated in four patients with multiple sclerosis and compared to α2-Macroglobulin from healthy controls. An impaired stability of α2-Macroglobulin from the multiple sclerosis patients was demonstrated as a spontaneous conversion to an electrophoretic‘‘fast” form of α2-Macroglobulin upon purification and storage, with a concomitant decrease in functional capacity to inhibit proteinases. The ability to form complexes with proteinases was significantly reduced in α2-Macroglobulin purified from the multiple sclerosis patients. The aberrant molecular arrangements of the protein were not due to proteinase cleavages in the bait regions of α2-Macroglobulin, as demonstrated by gel electrophoresis and protein sequencing. The number of functional thiol esters, however, was reduced in α2-Macroglobulin purified from the multiple sclerosis patients, an observation compatible with the impaired proteinase binding property. Furthermore, differences in isoelectric points were observed between α2-Macroglobulin from the multiple sclerosis patients and α2-Macroglobulin from healthy controls. The results suggest that aberrant forms of α2-Macroglobulin may be present in patients with multiple sclerosis.
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Immunochemical aberrations of alpha2-Macroglobulin purified from a patient with multiple sclerosis.
Acta neurologica Scandinavica, 2000Co-Authors: Martin Gunnarsson, Torgny Stigbrand, Poul Erik Hyldgaard JensenAbstract:We report on a patient with primary progressive multiple sclerosis who was found to present alpha2-Macroglobulin with aberrant immunochemical properties. alpha2-Macroglobulin was purified from plasma and investigated in ELISA using highly conformation specific monoclonal antibodies. The purified alpha2-Macroglobulin displayed reactivity with antibodies specific for binding to native alpha2-Macroglobulin as well as with antibodies specific for binding to transformed alpha2-Macroglobulin. Furthermore, following methylamine treatment, alpha2-Macroglobulin from the multiple sclerosis patient presented a reduced shift in antibody reactivity from the native-specific to the transformed-specific antibodies, as compared to normal alpha2-Macroglobulin. In conclusion, the results suggest that alpha2-Macroglobulin from this multiple sclerosis patient presents conformational aberrations, which may have implications on the capacity to eliminate proteinases from the systemic circulation.
Martin Gunnarsson - One of the best experts on this subject based on the ideXlab platform.
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Aberrant forms of α2-Macroglobulin purified from patients with multiple sclerosis
Clinica Chimica Acta, 2000Co-Authors: Martin Gunnarsson, Torgny Stigbrand, Poul Erik Hyldgaard JensenAbstract:Abstract The biochemical properties of α2-Macroglobulin were investigated in four patients with multiple sclerosis and compared to α2-Macroglobulin from healthy controls. An impaired stability of α2-Macroglobulin from the multiple sclerosis patients was demonstrated as a spontaneous conversion to an electrophoretic‘‘fast” form of α2-Macroglobulin upon purification and storage, with a concomitant decrease in functional capacity to inhibit proteinases. The ability to form complexes with proteinases was significantly reduced in α2-Macroglobulin purified from the multiple sclerosis patients. The aberrant molecular arrangements of the protein were not due to proteinase cleavages in the bait regions of α2-Macroglobulin, as demonstrated by gel electrophoresis and protein sequencing. The number of functional thiol esters, however, was reduced in α2-Macroglobulin purified from the multiple sclerosis patients, an observation compatible with the impaired proteinase binding property. Furthermore, differences in isoelectric points were observed between α2-Macroglobulin from the multiple sclerosis patients and α2-Macroglobulin from healthy controls. The results suggest that aberrant forms of α2-Macroglobulin may be present in patients with multiple sclerosis.
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Immunochemical aberrations of alpha2-Macroglobulin purified from a patient with multiple sclerosis.
Acta neurologica Scandinavica, 2000Co-Authors: Martin Gunnarsson, Torgny Stigbrand, Poul Erik Hyldgaard JensenAbstract:We report on a patient with primary progressive multiple sclerosis who was found to present alpha2-Macroglobulin with aberrant immunochemical properties. alpha2-Macroglobulin was purified from plasma and investigated in ELISA using highly conformation specific monoclonal antibodies. The purified alpha2-Macroglobulin displayed reactivity with antibodies specific for binding to native alpha2-Macroglobulin as well as with antibodies specific for binding to transformed alpha2-Macroglobulin. Furthermore, following methylamine treatment, alpha2-Macroglobulin from the multiple sclerosis patient presented a reduced shift in antibody reactivity from the native-specific to the transformed-specific antibodies, as compared to normal alpha2-Macroglobulin. In conclusion, the results suggest that alpha2-Macroglobulin from this multiple sclerosis patient presents conformational aberrations, which may have implications on the capacity to eliminate proteinases from the systemic circulation.
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Binding of Soluble Myelin Basic Protein to Various Conformational Forms of α2-Macroglobulin
Archives of Biochemistry and Biophysics, 1998Co-Authors: Martin Gunnarsson, Poul Erik H. JensenAbstract:Abstract Myelin basic protein is known to be released into the circulation following traumatic injuries or demyelination within the central nervous system, resulting in the generation of potentially immunogenic myelin basic protein material. In this investigation we have studied the binding of bovine and human myelin basic protein to human α 2 -Macroglobulin, which was found to be the only major myelin basic protein-binding protein in human plasma. Myelin basic protein bound to all three conformational forms of α 2 -Macroglobulin studied, i.e., native α 2 -Macroglobulin, methylamine-treated α 2 -Macroglobulin, and chymotrypsin-treated α 2 -Macroglobulin. Zinc chloride (1 mM) or 1 mM iodoacetamide partly blocked the complex formation between myelin basic protein and α 2 -Macroglobulin, while 1 mM magnesium chloride, 1 mM calcium chloride, or 1 mM EDTA had no effect on binding. Chymotrypsin and trypsin can degrade myelin basic protein to fragments which do not bind to α 2 -Macroglobulin. However, when myelin basic protein was complexed with any of the conformational forms of α 2 -Macroglobulin, no significant release of Na[ 125 I]-labeled myelin basic protein occurred after proteinase treatment. The results suggest that binding of myelin basic protein to α 2 -Macroglobulin may protect extracellular compartments in vivo from immunogenic myelin basic protein fragments and α 2 -Macroglobulin may participate in the specific clearance of myelin basic protein from the circulation.
Jan J. Enghild - One of the best experts on this subject based on the ideXlab platform.
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activated human plasma carboxypeptidase b is retained in the blood by binding to alpha2 Macroglobulin and pregnancy zone protein
Journal of Biological Chemistry, 1996Co-Authors: Zuzana Valnickova, Salvatore V. Pizzo, I B Thogersen, Soren Christensen, Jan J. EnghildAbstract:Abstract A 66-kDa glycosylated carboxypeptidase, plasma pro-carboxypeptidase B (pro-plasma CPB), has recently been identified in human blood (Eaton, D. L., Malloy, B. E., Tsai, S. P., Henzel, W., and Drayna, D. (1991) J. Biol. Chem. 266, 21833-21838). The pro-enzyme binds to plasminogen and the active enzyme is specific for COOH-terminal Lys or Arg residues. These properties implicate a role in the fibrinolytic or coagulation system. However, we show that the molecular mass of the active plasma CPB is approximately 36 kDa, which is below the glomerular filtration limit. Since activated plasma CPB no longer binds plasminogen, the active enzyme may not be retained in the circulation. To investigate this, we performed plasma elimination studies in mice which showed that 125I-plasma CPB remains in the circulation despite its small size. Native polyacrylamide gel electrophoresis of blood samples removed from the mice revealed that plasma CPB migrated as a high molecular weight band. Similar bands were observed in vitro when 125I-plasma CPB was added to plasma from humans and other species. The plasma CPB-binding proteins were purified from human plasma and identified as α2-Macroglobulin (α2M) and pregnancy zone protein. Only the active enzyme bound to the two α-Macroglobulins, and the interaction was specific for α2M in its native conformation, but not its receptor recognized forms. The complex between human α2M and plasma CPB dissociated during SDS-polyacrylamide gel electrophoresis and transverse urea gel electrophoresis suggesting that the interaction was noncovalent and depended on the tertiary structure of the native α2M molecule. The catalytic activity of plasma CPB was not significantly affected by its binding to α2M. The specific binding of plasma CPB to α-Macroglobulins suggest that these proteins may function as a “shuttle” in vivo to modulate the clearance of plasma CPB from the circulatory system.
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activated human plasma carboxypeptidase b is retained in the blood by binding to alpha2 Macroglobulin and pregnancy zone protein
Journal of Biological Chemistry, 1996Co-Authors: Zuzana Valnickova, Salvatore V. Pizzo, I B Thogersen, Soren Christensen, Charleen T Chu, Jan J. EnghildAbstract:Abstract A 66-kDa glycosylated carboxypeptidase, plasma pro-carboxypeptidase B (pro-plasma CPB), has recently been identified in human blood (Eaton, D. L., Malloy, B. E., Tsai, S. P., Henzel, W., and Drayna, D. (1991) J. Biol. Chem. 266, 21833-21838). The pro-enzyme binds to plasminogen and the active enzyme is specific for COOH-terminal Lys or Arg residues. These properties implicate a role in the fibrinolytic or coagulation system. However, we show that the molecular mass of the active plasma CPB is approximately 36 kDa, which is below the glomerular filtration limit. Since activated plasma CPB no longer binds plasminogen, the active enzyme may not be retained in the circulation. To investigate this, we performed plasma elimination studies in mice which showed that 125I-plasma CPB remains in the circulation despite its small size. Native polyacrylamide gel electrophoresis of blood samples removed from the mice revealed that plasma CPB migrated as a high molecular weight band. Similar bands were observed in vitro when 125I-plasma CPB was added to plasma from humans and other species. The plasma CPB-binding proteins were purified from human plasma and identified as α2-Macroglobulin (α2M) and pregnancy zone protein. Only the active enzyme bound to the two α-Macroglobulins, and the interaction was specific for α2M in its native conformation, but not its receptor recognized forms. The complex between human α2M and plasma CPB dissociated during SDS-polyacrylamide gel electrophoresis and transverse urea gel electrophoresis suggesting that the interaction was noncovalent and depended on the tertiary structure of the native α2M molecule. The catalytic activity of plasma CPB was not significantly affected by its binding to α2M. The specific binding of plasma CPB to α-Macroglobulins suggest that these proteins may function as a “shuttle” in vivo to modulate the clearance of plasma CPB from the circulatory system.
Torgny Stigbrand - One of the best experts on this subject based on the ideXlab platform.
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Proteolytic hydrolysis and purification of the LRP/alfa-2-Macroglobulin receptor domain from α-Macroglobulins
Protein expression and purification, 2006Co-Authors: Daniel Iván Barrera, Luisa M. Matheus, Torgny Stigbrand, Luis F ArbeláezAbstract:A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-Macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-Macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-Macroglobulin-proteinases complexes from the circulation by the LRP/receptor.
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Aberrant forms of α2-Macroglobulin purified from patients with multiple sclerosis
Clinica Chimica Acta, 2000Co-Authors: Martin Gunnarsson, Torgny Stigbrand, Poul Erik Hyldgaard JensenAbstract:Abstract The biochemical properties of α2-Macroglobulin were investigated in four patients with multiple sclerosis and compared to α2-Macroglobulin from healthy controls. An impaired stability of α2-Macroglobulin from the multiple sclerosis patients was demonstrated as a spontaneous conversion to an electrophoretic‘‘fast” form of α2-Macroglobulin upon purification and storage, with a concomitant decrease in functional capacity to inhibit proteinases. The ability to form complexes with proteinases was significantly reduced in α2-Macroglobulin purified from the multiple sclerosis patients. The aberrant molecular arrangements of the protein were not due to proteinase cleavages in the bait regions of α2-Macroglobulin, as demonstrated by gel electrophoresis and protein sequencing. The number of functional thiol esters, however, was reduced in α2-Macroglobulin purified from the multiple sclerosis patients, an observation compatible with the impaired proteinase binding property. Furthermore, differences in isoelectric points were observed between α2-Macroglobulin from the multiple sclerosis patients and α2-Macroglobulin from healthy controls. The results suggest that aberrant forms of α2-Macroglobulin may be present in patients with multiple sclerosis.
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Immunochemical aberrations of alpha2-Macroglobulin purified from a patient with multiple sclerosis.
Acta neurologica Scandinavica, 2000Co-Authors: Martin Gunnarsson, Torgny Stigbrand, Poul Erik Hyldgaard JensenAbstract:We report on a patient with primary progressive multiple sclerosis who was found to present alpha2-Macroglobulin with aberrant immunochemical properties. alpha2-Macroglobulin was purified from plasma and investigated in ELISA using highly conformation specific monoclonal antibodies. The purified alpha2-Macroglobulin displayed reactivity with antibodies specific for binding to native alpha2-Macroglobulin as well as with antibodies specific for binding to transformed alpha2-Macroglobulin. Furthermore, following methylamine treatment, alpha2-Macroglobulin from the multiple sclerosis patient presented a reduced shift in antibody reactivity from the native-specific to the transformed-specific antibodies, as compared to normal alpha2-Macroglobulin. In conclusion, the results suggest that alpha2-Macroglobulin from this multiple sclerosis patient presents conformational aberrations, which may have implications on the capacity to eliminate proteinases from the systemic circulation.
