[Studies on human alpha-2 macroglobulin structure and its complexes with proteases, using polyacrylamide gel electrophoresis].
Pure alpha2M is prepared with fresh plasma as starting material, to prevent the interaction of alpha2M from proteolytic enzymes of plasma such as thrombin, plasmin and kallikrein. During the purification steps, polybrene and aprotin are used as inhibitors and plasminogen is absorbed onto bentonite. When alpha 2M is submitted to polyacrylamide gel electrophoresis (PAA) containing 0.1% SDS, a complete dissociation in two half-molecules of MW 380,000 occurs. When alpha2M is incubated in 1% SDS and 1% beta-mercaptoethanol as reducing agent, only one component of MW 190,000 is observed in PAA-SDS. This experiments show that the alpha2M molecule consist of two symetric halves of same MW (380,000) linked by non covalent bonds. Each two-half-molecules is made of two polypeptides chains MW 190,000 linked by disulfide bonds. Thus alpha2M molecule contains four polypeptides chains having a same MW. The same techniques were applied to the study of alaph2M proteinases complexes. Three different proteinases (plasmin, trypsin and papain) were used in these experiments. Trypsin and papain are commercialy available. Plasminogen was obtained by affinity chromatography and activated into plasmin by insoluble streptokinase fixed on PAB cellulose.
D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000032:analysis*
D000511:alpha-Macroglobulins; D012967:Sodium Dodecyl Sulfate; D010447:Peptide Hydrolases; D014357:Trypsin; D005341:Fibrinolysin
Apport de l'électrophorèse en gel de polyacrylamide - SDS à l'étude de la structure de l'alpha-2 macroglobuline humaine et de ses complexes avec les protéinases