The role of His(143) for the pH-dependent stability of plasminogen activator inhibitor-1.
Using site-directed mutagenesis, His(143) on the alpha-helix F of PAI-1 was substituted by Lys, Asp, Phe and Thr, respectively. The generated single-site changed plasminogen activator inhibitor-1 (PAI-1) mutants were expressed in Escherichia coli and purified by heparin-Sepharose and anhydrotrypsin agarose chromatographies. When compared with wild-type (wtPAI-1), the PAI-1 mutants His143Asp and His143Phe had shorter half-lives at pH 7.5 (1.1 and 1.4 h, respectively, vs. 2 h for wtPAI-1). They also exhibited less pH dependency of their stability, with half-lives at pH 5.5 of 2.5 and 2.2 h, respectively, vs. 18 h for wtPAI-1. However, the PAI-1 mutants His143Lys and His143Thr had similar properties as wtPAI-1 in this respect. In conclusion, our results suggest that His(143) in one way or another might be involved in the pH-dependent stability of PAI-1. However, it seems that the protonation of this particular residue is of less importance. The PAI-1 mutants His143Asp and His143Phe only displayed about 20% of the specific activity obtained for wtPAI-1, because they, to a large extent, act as substrates for tissue-type plasminogen activator.
Sui
G C
GC
Department of Laboratory Medicine, Division of Clinical Chemistry and Blood Coagulation, Karolinska Hospital, Karolinska Institute S-171 76, Stockholm, Sweden.
Mångs
H
H
Wiman
B
B
D004355:Drug Stability; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors
D017395:Plasminogen Activator Inhibitor 1; D015842:Serine Proteinase Inhibitors; D006639:Histidine; D010959:Tissue Plasminogen Activator