EXPRESSION, PURIFICATION AND INHIBITORY PROPERTIES OF HUMAN PROTEINASE-INHIBITOR-8

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian express ion vector and expressed in baby hamster kidney cells, Initial studies indicated that PI8 was able to inhibit the amidolytic activity of try psin and form an SDS-stable similar to 67-kDa complex with human throm bin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861], I n the present study, we have expressed recombinant PI8 in the methylot ropic yeast Pichia pastoris, purified the inhibitor to homogeneity, an d investigated its ability to inhibit a variety of proteinases. PI8 in hibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endopro teinase subtilisin A through different mechanisms but failed to inhibi t the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsi n in a purely competitive manner, with an equilibrium inhibition const ant (K-i) of less than 3.8 nM. The interaction between PI8 and thrombi n occurred with a second-order association rate constant (k(assoc)) of 1.0 x 10(5) M-1 s(-1) and a K-i of 350 pM. A slow-binding kinetics ap proach was used to determine the kinetic constants for the interaction s of PIS with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a k(assoc) of 7.5 x 10(4) M-1 s(-1) and an o verall K-i of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a k(assoc) of 1.16 x 10(6) M-1 s(-1) and a n overall K-i of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evoluti onary precursor to the intracellular mammalian dibasic processing endo proteinases.