A functionally active heavy chain derived from human high molecular weight urokinase.

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Human high molecular weight urokinase (HMW-UK) is a serine protease and a plasminogen activator.
• HMW-UK plays a crucial role in fibrinolysis, the process of breaking down blood clots.
• Understanding the structure-function relationship of HMW-UK is vital for developing targeted therapies.

Purpose of the Study:

• To isolate and characterize the functionally active heavy chain of human HMW-UK.
• To compare the biochemical and kinetic properties of the isolated heavy chain with low molecular weight urokinase (LMW-UK).
• To elucidate the role of the heavy chain in urokinase's enzymatic activity.

Main Methods:

• Limited reduction of HMW-UK with 2-mercaptoethanol followed by carboxymethylation.
• Affinity chromatography using [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose for heavy chain isolation.
• Gel filtration (Sephadex G-100) and SDS-PAGE for purification and molecular weight determination.
• Enzyme activity assays using specific chromogenic substrates and active site titration.

Main Results:

• A functionally active heavy chain (approximately 80% of original activity) and an inactive light chain were obtained, linked by a disulfide bond.
• The isolated heavy chain exhibited similar molecular weight, specific activity, and amino acid composition to LMW-UK.
• Kinetic parameters for plasminogen activation and amidase activity of the heavy chain were comparable to LMW-UK.
• The NH2-terminal residue of the active heavy chain was identified as lysine, consistent with LMW-UK.

Conclusions:

• The active heavy chain of HMW-UK is responsible for its enzymatic activity and shares functional similarities with LMW-UK.
• The isolation and characterization of the active heavy chain provide insights into the structure-function relationship of urokinase.
• These findings support the concept that LMW-UK is derived from HMW-UK and highlight the importance of the heavy chain in fibrinolytic activity.