Bovine enterokinase. Purification, specificity, and some molecular properties

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Abstract
Enterokinase (EC 3.4.4.8) was isolated from the contents of bovine duodena and purified 1200-fold in 41% yield. The isolation procedure employed DEAE chromatography, affinity chromatography on immobilized p-aminobenzamidine and gel filtration. The resultant pure enzyme exhibits a single band on sodium dodecyl sulfate gel electrophoresis corresponding to a MW of 145,000. Two chains in the molecule, connected by disulfide linkages, have MW of 57,000 and 82,000, respectively. The purified enzyme exhibits a restricted specificity which is directed toward the polyanionic amino acid sequence in the activation peptide of the zymogen substrate. Of the zymogens of the serine proteases tested, including several of those involved in blood coagulation, only native and guanidinated trypsinogen are activated by enterokinase, whereas acetylated trypsinogen is not. Partial heat denaturation of bovine enterokinase causes a differential response toward the activation of trypsinogen and the hydrolysis of benzoyl-L-arginine ethyl ester (BzArgOEt), further suggesting that secondary sites are important in the binding of trypsinogen. A sensitive assay for enterokinase (nanomole range) was developed using tritiated BzArgOEt.

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