BOVINE ENTEROKINASE: MEMBRANE-BOUND PROPERTIES AND CHARACTERIZATION OF THE ISOLATED CATALYTIC SUBUNIT

PETER FONSECA, Purdue University

Abstract

Bovine enterokinase (enteropeptidase, E.C.3.4.21.9) was found to be an integral membrane protein of the brush border of the small intestine. Enterokinase was incorporated into vesicles reconstituted from a soybean phospholipid mixture by a thin film hydration procedure. The properties of the enzyme in the vesicles were compared with those of alkaline phosphatase which was also present (used as a brush border membrane marker protein) and with the enzyme activities in vesicles prepared from brush border membranes. The reconstituted system was stable to solutions of high ionic strength. The enzymes were released from the membranes with detergent and on incubation with papain. The papain-released enterokinase was fully active, but could not be reincorporated into phospholipid vesicles. Enterokinase activity was also found associated with a viscous fluid lining the small intestinal wall. This fluid was an ideal starting source for the preparation of the enzyme and resulted in 35 to 50 mg of enterokinase recovered from 4 kg of mucosal fluid in yields of 30-35%. The properties were identical to the mucosal cell enzyme and could also be reconstituted into a phospholipid membrane. The catalytic subunit of enterokinase was obtained by the selective reduction of native enterokinase with dithioerythritol and separated from the heavy chain. The catalytic subunit had three half-cystines and the heavy chain had nine half-cystines. The activity of the catalytic subunit towards small synthetic substrates was similar to the native enzyme and both were inhibited by Kunitz pancreatic trypsin inhibitor. The catalytic subunit was also inhibited by soybean trypsin inhibitor whereas the native molecule was not. The catalytic chain retained its specificity towards S-carboxymethylated trypsinogen and S-carboxymethylated bovine serum albumin, although the rates of hydrolysis were slower. The catalytic subunit could not be incorporated into phospholipid vesicles. It is concluded that the catalytic subunit is responsible for specificity and catalysis. The heavy subunit provides the hydrophobic anchor for attachment to the membrane.

Degree

Ph.D.

Subject Area

Biochemistry

COinS