Additional observations on cholecystokinin and the vasoactive intestinal polypeptide

Area of Science:

• Biochemistry
• Peptide Chemistry

Background:

• Oxidation can inactivate peptides such as cholecystokinin (CCK) during isolation and bioassays.
• Methionine sulfoxide reduction is key to restoring peptide function.

Purpose of the Study:

• To investigate the reactivation of oxidized peptides, specifically CCK, VIP, and tetragastrin.
• To explore the potential of N-methylmercaptoacetamide as a reducing agent for peptide restoration.
• To identify enzymatic pathways involved in CCK reactivation.

Main Methods:

• Reduction of oxidized peptides (CCK, VIP, tetragastrin) with N-methylmercaptoacetamide at room temperature.
• Thin layer chromatography on silica gel to assess peptide integrity.
• Enzymatic assays to investigate CCK reactivation by liver cytosol and thioredoxin.
• Analysis of CCK cleavage by enterokinase and dipeptidyl amino-peptidase I.

Main Results:

• N-methylmercaptoacetamide showed promising but not unequivocal success in reactivating oxidized CCK.
• Oxidized VIP and tetragastrin were reconverted to their preoxidation states using N-methylmercaptoacetamide.
• Evidence suggests a rat liver cytosol enzyme catalyzes CCK reductive reactivation using reduced thioredoxin.
• Enterokinase cleaves CCK, releasing smaller fragments, and dipeptidyl amino-peptidase I converts 39-CCK to 33-CCK.

Conclusions:

• N-methylmercaptoacetamide is a potential agent for peptide reactivation, particularly for VIP and tetragastrin.
• A liver enzyme system involving thioredoxin likely plays a role in CCK reactivation.
• Specific proteases like enterokinase and dipeptidyl amino-peptidase I are involved in CCK processing.