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Structure and function of microplasminogen: reconstitution of microplasminogen and microplasmin from isolated
T de los Santos1, J Wang, E Reich
1Department of Pharmacological Sciences, State University of New York at Stony Brook 11794-8651, USA.
Abstract:
We describe limited chemical proteolysis of microplasminogen/microplasmin (mPlg/mPlm) and their reconstitution from isolated fragments. A V141-->M141 substitution in methionineless human mPlg/mPlm allowed the protein(s) to be cleaved in CNBr/formic acid. The resulting two fragments (141 and 118 residues, respectively), each internally disulfide bonded, were separated by preparative non-reducing gradient SDS-PAGE, and could then be mixed to reconstitute the characteristic mPlg/mPlm, including their activation by urokinase (uPA) and streptokinase (SK), and inhibition by macromolecular inhibitors. The isolated larger, N-terminal fragment, which contains the mPlg activation site in a normal disulfide configuration, was not cleaved by uPA in the absence of its smaller C-terminal companion, showing that the linear amino acid sequence is not by itself sufficient to confer substrate character, even when its conformation is constrained by the disulfide structure.
Insights
Researchers chemically cleaved and reconstituted microplasminogen/microplasmin (mPlg/mPlm) fragments. The N-terminal fragment alone did not activate with urokinase (uPA), indicating sequence alone is insufficient for substrate character.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Microplasminogen (mPlg) and microplasmin (mPlm) are key players in fibrinolysis.
- Understanding their structure-function relationship is crucial for therapeutic development.
Purpose of the Study:
- To investigate the role of specific protein fragments in mPlg/mPlm activation and function.
- To determine if linear amino acid sequence or conformational structure dictates substrate character.
Main Methods:
- Limited chemical proteolysis of methionineless human mPlg/mPlm using CNBr/formic acid.
- Separation of resulting fragments (141 and 118 residues) via non-reducing gradient SDS-PAGE.
- Reconstitution of mPlg/mPlm from isolated fragments and assessment of activation by urokinase (uPA) and streptokinase (SK), and inhibition by macromolecular inhibitors.
Main Results:
- Successful cleavage and isolation of two disulfide-bonded fragments from mPlg/mPlm.
- Reconstituted mPlg/mPlm exhibited characteristic activation and inhibition profiles.
- The isolated N-terminal fragment, despite containing the activation site, was not activated by uPA without the C-terminal fragment.
Conclusions:
- Protein fragmentation and reconstitution confirm the importance of specific domains for mPlg/mPlm activity.
- Substrate character is not solely determined by the amino acid sequence or disulfide bonding, but requires the presence of both fragments for proper function.