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Isolation and characterization of microplasminogen. A low molecular weight form of plasminogen
1Biochemistry Department, Medical College, National Cheng-Kung University, Tainan, Taiwan, Republic of China.
Abstract:
A functionally active human microplasminogen without kringle structures was produced by incubation of plasminogen with urokinase-free plasmin at an alkaline pH. The microplasminogen was purified by affinity chromatography on lysine- and soybean trypsin inhibitor-Sepharose and by chromofocusing. Human plasminogen is specifically cleaved at Arg529-Lys530 by plasmin to form microplasminogen, which consists of a single polypeptide of 261 residues from the COOH-terminal portion of native plasminogen. It has an Mr of 28,617, calculated from the sequence, which is consistent with the molecular weight determined by sodium dodecyl sulfate gel electrophoresis. Microplasminogen is a slightly basic protein and is eluted from a chromofocusing column at pH 8.3. It can be activated by urokinase and streptokinase to a catalytically active microplasmin. The specific amidolytic activity of microplasmin is about three times higher than Lys77-plasmin on a weight basis and is about the same on a molar basis. The activation of microplasminogen by streptokinase is slower than that of either Glu-plasminogen or Lys77-plasminogen. On the other hand, the activation of microplasminogen by urokinase is faster than that of either of the latter. The Arg560-Val561 bond is cleaved during activation of both microplasminogen and native plasminogen.
Insights
A novel human microplasminogen, lacking kringle structures, was synthesized and purified. This microplasminogen demonstrates enhanced activation by urokinase, yielding a more active microplasmin.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Plasminogen is a key protein in the fibrinolytic system.
- Kringle structures are important domains in native plasminogen.
- Understanding plasminogen variants can reveal insights into fibrinolysis regulation.
Purpose of the Study:
- To produce and characterize a functionally active human microplasminogen.
- To investigate the activation kinetics and enzymatic activity of microplasmin.
Main Methods:
- Incubation of plasminogen with urokinase-free plasmin at alkaline pH.
- Purification using affinity chromatography and chromofocusing.
- Characterization by SDS-PAGE and sequence analysis.
Main Results:
- Successfully produced and purified human microplasminogen (261 residues, Mr 28,617).
- Microplasminogen is activated by urokinase and streptokinase to form microplasmin.
- Microplasmin exhibits higher specific amidolytic activity than Lys77-plasmin.
- Urokinase activation of microplasminogen is faster than native plasminogen.
Conclusions:
- Microplasminogen represents a distinct functional entity of plasminogen.
- The absence of kringle structures influences activation kinetics and enzymatic efficiency.
- Microplasminogen and its derived microplasmin are valuable tools for studying fibrinolysis.