Importance of intramolecular interactions in the control of the fibrin affinity and activation of human plasminogen.
Incubation of human Glu-plasminogen with 1,5-difluoro-2,4-dinitrobenzene leads to the specific intra-molecular cross-linking of the kringle 1+2+3 region and the light (B) chain region of plasminogen. This cross-link was found to prevent the conformational change which is induced in Glu-plasminogen by lysine analogues or by proteolytic removal of the NH2-terminal peptide. Our results suggest that the cross-link freezes the closed conformation of Glu-plasminogen, and it seems likely that the transition to the loose conformer requires separation of the kringle 1+2+3 region from the light (B) chain portion. The change in the relative position of these regions during the conformational change in plasminogen is also indicated by our observation that the rate of formation of the intramolecular cross-link is significantly decreased when transition to the loose conformer is induced either by saturation of the lysine-binding sites or by conversion to Lys-plasminogen. Cross-linked Glu-plasminogen is slowly activated by urokinase and melanoma tissue plasminogen activator, but in contrast with uncross-linked Glu-plasminogen conversion to Lys-plasminogen or saturation of lysine-binding sites with ligand does not increase the rate of activation because the cross-link prevents transition to the loose conformer which is susceptible to activation. The fibrin affinity of cross-linked Glu-plasminogen is practically identical with that of Glu-plasminogen. As in the case of uncross-linked Glu-plasminogen, removal of the NH2-terminal peptide causes a marked increase in fibrin affinity although the resulting cross-linked Lys-plasminogen is fixed in the closed conformation. This result suggests that the NH2-terminal peptide inhibits binding of plasminogen to fibrin by direct interaction with the fibrin-binding site, and the conformational change that normally accompanies its removal is not a prerequisite of strong binding.
D003432:Cross-Linking Reagents; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010958:Plasminogen / Q000378:metabolism*; D011487:Protein Conformation
D003432:Cross-Linking Reagents; D005337:Fibrin; D010958:Plasminogen; D004139:Dinitrofluorobenzene; C002041:1,5-difluoro-2,4-dinitrobenzene