Protransglutaminase E from guinea pig skin. Isolation and partial characterization.

Area of Science:

• Biochemistry
• Dermatology
• Enzymology

Background:

• Epidermal transglutaminase E plays a crucial role in skin structure and barrier function.
• The enzyme exists in a zymogen form, protransglutaminase E, in the skin.
• Understanding the activation process of protransglutaminase E is key to comprehending epidermal homeostasis.

Purpose of the Study:

• To isolate and characterize protransglutaminase E from adult guinea pig skin.
• To investigate the activation mechanism of protransglutaminase E into its active form.
• To elucidate the subunit composition and structural features of the active enzyme.

Main Methods:

• Isolation of protransglutaminase E from guinea pig skin.
• Molecular weight determination using sedimentation equilibrium, exclusion chromatography, and SDS-PAGE.
• Enzyme activation studies using various proteases (dispase, proteinase K, trypsin, thrombin).
• Analysis of the active enzyme's subunit dissociation under denaturing conditions.

Main Results:

• Protransglutaminase E was isolated and characterized with a molecular weight of approximately 77,800 Da.
• Activation by proteases yielded active transglutaminase E.
• The active enzyme exists as a non-covalently associated dimer of 50,000 Da and 27,000 Da fragments under denaturing conditions.
• The catalytically essential thiol group resides in the 50,000 Da fragment, while the 27,000 Da fragment has an unmasked N-terminus.

Conclusions:

• A model for protransglutaminase E activation involving noncovalent dissociation into functional fragments is proposed.
• The 50,000 Da fragment contains the active site, and the 27,000 Da fragment may regulate activation.
• Further studies are needed to determine the role of the noncatalytic fragment in enzyme function.