Purification and characterization of a novel, oligomeric, plasminogen kringle 4 binding protein from human plasma: tetranectin.
Purification of alpha 2-plasmin inhibitor (alpha 2PI) from human plasma by affinity chromatography on plasminogen-Sepharose resulted in copurification of a contaminating protein with Mr 17,000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This contaminating protein could not be removed from the purified alpha 2-PI preparation by several types of gel chromatography applied. The use of the kringle 1-3 part of plasminogen, K(1 + 2 + 3), bound to Sepharose for affinity chromatography, instead of plasminogen-Sepharose, resulted in an alpha 2PI preparation without this contaminant. The contaminating protein was found to interact specifically with the kringle 4 part of plasminogen (K4) and not with K(1 + 2 + 3) or miniplasminogen. The K4-binding protein was purified by ammonium sulphate precipitation, affinity chromatography on K4-Sepharose, ion-exchange chromatography and gel filtration on AcA 34. The relative molecular mass of the protein (Mr 68 000) was estimated by gel filtration. This suggests a tetrameric protein composed of four subunits (Mr 17,000), that are dissociated by 1% sodium dodecyl sulphate. Dissociation into subunits was also demonstrated by gel filtration in the presence of 6 M guanidine hydrochloride. A specific antibody was raised in rabbits against the purified protein and this antibody was shown not to react with any known fibrinolytic components. The pI of the K4-binding protein was found to be 5.8. The first three N-terminal amino acids were determined to be Glu-Pro-Pro. The concentration of the protein in plasma was estimated to be 0.20 +/- 0.03 microM (15 +/- 2 mg/l). The electrophoretic mobility of the K4-binding protein was shown by crossed immunoelectrophoresis to be influenced by the presence of Ca2+, EDTA and heparin. The protein was found to enhance plasminogen activation catalyzed by tissue-type plasminogen activator (t-PA) in the presence of poly(D-lysine). The protein appeared to be a novel plasma protein tentatively called 'tetranectin'.
D001798:Blood Proteins / Q000032:analysis / Q000302:isolation & purification*; D002118:Calcium; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D005342:Fibrinolysis; D006493:Heparin; D006801:Humans; D007122:Immunoelectrophoresis / Q000379:methods; D007525:Isoelectric Focusing; D037181:Lectins, C-Type; D010446:Peptide Fragments / Q000097:blood*; D010958:Plasminogen / Q000378:metabolism*; D010960:Plasminogen Activators; D011107:Polylysine / Q000494:pharmacology; D011485:Protein Binding; D000979:alpha-2-Antiplasmin / Q000378:metabolism
D001798:Blood Proteins; D037181:Lectins, C-Type; D010446:Peptide Fragments; D000979:alpha-2-Antiplasmin; C048648:tetranectin; D011107:Polylysine; D010958:Plasminogen; D006493:Heparin; D004492:Edetic Acid; D010960:Plasminogen Activators; D002118:Calcium