Bovine enterokinase. Purification, specificity, and some molecular properties

Area of Science:

• Biochemistry
• Enzymology
• Protease research

Background:

• Enterokinase is a critical enzyme involved in the activation of digestive zymogens.
• Understanding enterokinase's properties and specificity is essential for its biotechnological applications.
• Previous purification methods were less efficient, limiting detailed study.

Purpose of the Study:

• To isolate and highly purify bovine enterokinase from duodenal contents.
• To characterize the purified enzyme's molecular weight, subunit composition, and specificity.
• To develop a sensitive assay for quantifying enterokinase activity.

Main Methods:

• Purification involved DEAE chromatography, affinity chromatography (p-aminobenzamidine), and gel filtration.
• Enzyme purity and molecular weight were assessed using SDS-PAGE.
• Enzyme specificity was tested against various serine protease zymogens and a synthetic substrate (BZArgOEt).

Main Results:

• Enterokinase was purified 1200-fold with 41% yield, showing a single band (145,000 MW) on SDS-PAGE.
• The enzyme consists of two disulfide-linked chains (57,000 and 82,000 MW).
• Purified enterokinase specifically activated native and guanidinated trypsinogen, but not acetylated trypsinogen, indicating a specific recognition site. Heat denaturation studies suggested secondary binding sites.

Conclusions:

• A highly purified and active form of bovine enterokinase was obtained.
• The enzyme exhibits high specificity for activating trypsinogen, primarily through its polyanionic activation peptide sequence.
• A sensitive nanomole-range assay for enterokinase was successfully developed using tritiated BZArgOEt.