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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, I B Thøgersen, Salvatore V. Pizzo, 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.
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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, I B Thøgersen, Salvatore V. Pizzo, 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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Purification and characterization of an alpha-macroglobulin proteinase inhibitor from the mollusc Octopus vulgaris.
Biochemical Journal, 1992Co-Authors: I B Thøgersen, Guy S. Salvesen, F H Brucato, Salvatore V. Pizzo, Jan J. EnghildAbstract:The cell-free haemolymph of the mollusc Octopus vulgaris inhibited the proteolytic activity of the thermolysin against the high-molecular-mass substrate hide powder azure. The purified inhibitor was a glycoprotein composed of two identical 180 kDa disulphide-linked subunits. In addition to the inhibition of the metalloproteinase thermolysin, the protein inhibited the serine proteinases human neutrophil elastase, pig pancreatic elastase, bovine chymotrypsin, bovine trypsin and the cysteine proteinase papain. A fraction of the proteinase-inhibitor complex resisted dissociation after denaturation indicating that some of the proteinase molecules became covalently bound. The nucleophile beta-aminopropionitrile decreased the covalent binding of proteinases to the Octopus vulgaris protein, suggesting that this interaction is mediated by an internal thiol ester; the reactivity and the amino acid sequence flanking the reactive residues of the putative thiol ester were consistent with this hypothesis. Bound trypsin remained active against the low-molecular-mass chromatogenic substrate H-D-Pro-Phe-Arg p-nitroanilide and was protected from inhibition by active-site-directed protein inhibitors of trypsin; however, the bound trypsin was readily inhibited by small synthetic inhibitors. This indicates that the inhibition of proteinases is accomplished by steric hindrance. The proteinase-inhibitory activity of this protein is characteristic of inhibition by mammalian alpha-Macroglobulins and the presence of a putative thiol ester suggests that the Octopus vulgaris proteinase inhibitor is a homologue of human alpha 2-macroglobulin.
I B Thøgersen - 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, I B Thøgersen, Salvatore V. Pizzo, 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.
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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, I B Thøgersen, Salvatore V. Pizzo, 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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Purification and characterization of an alpha-macroglobulin proteinase inhibitor from the mollusc Octopus vulgaris.
Biochemical Journal, 1992Co-Authors: I B Thøgersen, Guy S. Salvesen, F H Brucato, Salvatore V. Pizzo, Jan J. EnghildAbstract:The cell-free haemolymph of the mollusc Octopus vulgaris inhibited the proteolytic activity of the thermolysin against the high-molecular-mass substrate hide powder azure. The purified inhibitor was a glycoprotein composed of two identical 180 kDa disulphide-linked subunits. In addition to the inhibition of the metalloproteinase thermolysin, the protein inhibited the serine proteinases human neutrophil elastase, pig pancreatic elastase, bovine chymotrypsin, bovine trypsin and the cysteine proteinase papain. A fraction of the proteinase-inhibitor complex resisted dissociation after denaturation indicating that some of the proteinase molecules became covalently bound. The nucleophile beta-aminopropionitrile decreased the covalent binding of proteinases to the Octopus vulgaris protein, suggesting that this interaction is mediated by an internal thiol ester; the reactivity and the amino acid sequence flanking the reactive residues of the putative thiol ester were consistent with this hypothesis. Bound trypsin remained active against the low-molecular-mass chromatogenic substrate H-D-Pro-Phe-Arg p-nitroanilide and was protected from inhibition by active-site-directed protein inhibitors of trypsin; however, the bound trypsin was readily inhibited by small synthetic inhibitors. This indicates that the inhibition of proteinases is accomplished by steric hindrance. The proteinase-inhibitory activity of this protein is characteristic of inhibition by mammalian alpha-Macroglobulins and the presence of a putative thiol ester suggests that the Octopus vulgaris proteinase inhibitor is a homologue of human alpha 2-macroglobulin.
Salvatore V. Pizzo - 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, I B Thøgersen, Salvatore V. Pizzo, 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.
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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, I B Thøgersen, Salvatore V. Pizzo, 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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Purification and characterization of an alpha-macroglobulin proteinase inhibitor from the mollusc Octopus vulgaris.
Biochemical Journal, 1992Co-Authors: I B Thøgersen, Guy S. Salvesen, F H Brucato, Salvatore V. Pizzo, Jan J. EnghildAbstract:The cell-free haemolymph of the mollusc Octopus vulgaris inhibited the proteolytic activity of the thermolysin against the high-molecular-mass substrate hide powder azure. The purified inhibitor was a glycoprotein composed of two identical 180 kDa disulphide-linked subunits. In addition to the inhibition of the metalloproteinase thermolysin, the protein inhibited the serine proteinases human neutrophil elastase, pig pancreatic elastase, bovine chymotrypsin, bovine trypsin and the cysteine proteinase papain. A fraction of the proteinase-inhibitor complex resisted dissociation after denaturation indicating that some of the proteinase molecules became covalently bound. The nucleophile beta-aminopropionitrile decreased the covalent binding of proteinases to the Octopus vulgaris protein, suggesting that this interaction is mediated by an internal thiol ester; the reactivity and the amino acid sequence flanking the reactive residues of the putative thiol ester were consistent with this hypothesis. Bound trypsin remained active against the low-molecular-mass chromatogenic substrate H-D-Pro-Phe-Arg p-nitroanilide and was protected from inhibition by active-site-directed protein inhibitors of trypsin; however, the bound trypsin was readily inhibited by small synthetic inhibitors. This indicates that the inhibition of proteinases is accomplished by steric hindrance. The proteinase-inhibitory activity of this protein is characteristic of inhibition by mammalian alpha-Macroglobulins and the presence of a putative thiol ester suggests that the Octopus vulgaris proteinase inhibitor is a homologue of human alpha 2-macroglobulin.
Fahim Halim Khan - One of the best experts on this subject based on the ideXlab platform.
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interaction of anti cancer drug cisplatin with major proteinase inhibitor alpha 2 macroglobulin biophysical and thermodynamic analysis
International Journal of Biological Macromolecules, 2018Co-Authors: Mohammad Khalid Zia, Haseeb Ahsan, Syed Saqib Ali, Tooba Siddiqui, Fahim Halim KhanAbstract:Alpha-2-macroglobulin is a multifunctional, highly abundant, plasma protein which reacts with a wide variety of molecules and drugs including cisplatin. Cisplatin is commonly used anticancer drug widely used for treatment of testicular, bladder, ovarian, head and neck, lung and cervical cancers. This study is designed to examine the interaction of cisplatin with human alpha-2-macroglobulin through various biophysical techniques and drug binding through molecular modeling. Cisplatin alters the function of alpha-2-macroglobulin and the thiolesters are most likely the reactive sites for cisplatin. Our result suggests that cisplatin decreases the antiproteolytic potential and causes structural and functional change in human alpha-2-macroglobulin as evident by absorption and fluorescence spectroscopy. Change in secondary structure of alpha-2-macroglobulin was confirmed by CD and FTIR. Thermodynamics parameters such as entropy (ΔS), enthalpy (ΔH) and Gibb's free energy changes (ΔG) along with number of binding sites (N) of alpha-2-macroglobulin-cisplatin binding in solutions were determined by isothermal titration calorimetry (ITC). It was found that binding of cisplatin with alpha-2-macroglobulin was exothermic in nature. The interaction of drug with alpha-2-macroglobulin in the plasma could lead to structural alterations in the conformational status of alpha-2-macroglobulin resulting in its functional inactivation.
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α 2 macroglobulin a physiological guardian
Journal of Cellular Physiology, 2013Co-Authors: Ahmed Abdur Rehman, Haseeb Ahsan, Fahim Halim KhanAbstract:Alpha Macroglobulins are large glycoproteins which are present in the body fluids of both invertebrates and vertebrates. Alpha-2-macroglobulin (α2 M), a key member of alpha macroglobulin superfamily, is a high-molecular weight homotetrameric glycoprotein. α2 M has many diversified and complex functions, but it is primarily known by its ability to inhibit a broad spectrum of proteases without the direct blockage of the protease active site. α2 M is also known to be involved in the regulation, transport, and a host of other functions. For example, apart from inhibiting proteinases, it regulates binding of transferrin to its surface receptor, binds defensin and myelin basic protein, etc., binds several important cytokines, including basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and modify their biological activity. α2 M also binds a number of hormones and regulates their activity. α2 M is said to protect the body against various infections, and hence, can be used as a biomarker for the diagnosis and prognosis of a number of diseases. However, this multipurpose antiproteinse is not "fail safe" and could be damaged by reactive species generated endogenously or exogenously, leading to various pathophysiological conditions.
Barrera, Daniel Iván - 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 ?-Macroglobulins
'Elsevier BV', 2020Co-Authors: Barrera, Daniel Iván, Matheus, Luisa Marina, Stigbrand Torgny, Arbeláez, Luis FernandoAbstract:"A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two ?-Macroglobulins, ?2-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of ?30 kDa and the N-terminal sequences were determined to be SSTQDTV for ?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 ?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 ?-Macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate ?-macroglobulin-proteinases complexes from the circulation by the LRP/receptor. © 2006 Elsevier Inc. All rights reserved.
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Identificación electroforética de 2-macroglobulina en plasma de ovino de pelo (Ovis aries) y búfalo (Bubalus bubalis)
Universidad de Antioquia: Facultad de Ciencias Agrarias, 2007Co-Authors: Barrera, Daniel Iván, Giraldo, Jorge H., Duque, Carlos M., Arbeláez Ramírez, Luis F.Abstract:Blood plasma from six different non pregnant and pregnant species, including human blood plasma, was analyzed for detection of á2-Macroglobulin (á2-M). The tropical hair sheep (Ovis aries) and the buffalo (Bubalus bubalis) were studied for the first time in Colombia. The presence of the á2-M in plasma of all the species was demonstrated by SDS 7.5% PAGE as bands of 180 kDa as well as by non-denaturing 5% PAGE with bands of 720 kDa. The tetrameric form �¿2-M (tetramerica) and the pregnancy zone protein (PZP) (dimeric) purified at 98%, as well as its corresponding bans from human plasma were used as control. The N-terminal sequence of the band of 180 kDa in Tropical hair sheep plasma was very similar to the purified human �¿2-M. The results indicated the presence of �¿2-M in blood plasma of all the species tested, while the PZP was present only in the pregnant human plasma. Both human and bovine �¿2-M became activated with the fast form by reacting with Methylamine. This Fac. demonstrates the differences in the reactivity of the animal�fs �¿2-M with primary amine as compared with the human �¿2-M. It could be necessary to unify purification methods into one method for all species, so that the sensitive domain of the �¿-Macroglobulins (thiolester and bait region) receives the same treatment and grade of denaturation for all �¿2-M preparation.A traves del presente estudio se analizaron plasmas sanguineos de seis especies, incluyendo el humano tanto en estado gestante como no gestante, identificandose por primera vez en plasma, la glicoproteina �¿2-Macroglobulina (�¿2-M) de ovino de pelo (Ovis aries) y de bufalo (Bubalus bubalis). La presencia de esta proteina en el plasma sanguineo de todas las especies en estudio se demostro mediante electroforesis en gel de poliacrilamida usando sodio dodecilsulfato como agente denaturante (SDS PAGE) al 7.5% identificandose como bandas de 180 kDa y en forma no denaturante PAGE 5% como bandas de 720 kDa. Estas ultimas bandas fueron claramente intercambiables de la forma tetramerica a la forma monomerica en los ensayos electroforeticos. Como controles se usaron la �¿2-M (tetramerica) y la proteina de la zona de gestacion (PZP) (dimerica) purificadas a un 98%; asi como, las bandas de estas dos proteinas en el plasma humano. El analisis de la secuencia del dominio N-terminal de la (�¿2-M) de ovino de pelo, fue muy similar al de la proteina humana purificada. Tanto la �¿2-M humana como la bovina llegaron a ser activadas a la forma rapida por medio de la reaccion con metilamina. Lo anterior demuestra diferencias en la reactividad de las �¿2-M animales con la amina primaria cuando se comparan los resultados con la forma rapida de la �¿2-M humana. Sera necesario unificar los metodos de purificacion de esta proteina en todas las especies, de tal manera que los dominios sensibles de las �¿-macroglobulinas (tioester y region senuelo) tengan el mismo tratamiento y el mismo grado de desnaturalizacion para todas las preparaciones de �¿2-M
