Analysis of the interactions between streptokinase domains and human plasminogen.
The contrasting roles of streptokinase (SK) domains in binding human Glu1-plasminogen (Plg) have been studied using a set of proteolytic fragments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed using gel filtration chromatography and surface plasmon resonance. The latter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SK or SK fragments. Each of the SK fragments that contains domain B (fragments A2-B-C, A2-B, B-C, and B) binds Plg with similar affinity, at a level approximately 100- to 1,000-fold lower than intact SK. Experiments using 10 mM 6-aminohexanoic acid or 50 mM benzamidine demonstrate that either of these two lysine analogues abolishes interaction of domain B with Plg. Isolated domain C does not show detectable binding to Plg. Moreover, the additional presence of domain C within other SK fragments (B-C and A2-B-C) does not alter significantly their affinities for Plg. In addition, Plg-binding by a noncovalent complex of two SK fragments that contains domains A and B is similar to that of domain B. By contrast, species containing domain B and both domains A and C (intact SK and the two-chain complex A1 x A2-B-C) show a significantly higher affinity for Plg, which could not be completely inhibited by saturating amounts of 6-AHA. These results show that SK domain B interacts with Plg in a lysine-dependent manner and that although domains A and C do not appear independently to possess affinity for Plg, they function cooperatively to establish the additional interactions with Plg to form an efficient native-like Plg activator complex.
Conejero-Lara
F
F
Oxford Centre for Molecular Sciences and New Chemistry Laboratory, University of Oxford, United Kingdom.
Parrado
J
J
Azuaga
A I
AI
Dobson
C M
CM
Ponting
C P
CP
D015119:Aminocaproic Acid / Q000378:metabolism; D001550:Benzamidines / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive; D002850:Chromatography, Gel; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D013300:Streptokinase / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D001550:Benzamidines; D010446:Peptide Fragments; D010958:Plasminogen; D013300:Streptokinase; C032157:benzamidine; D015119:Aminocaproic Acid