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 expression vector and expressed in baby hamster kidney cells, Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable similar to 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861], In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (K-i) of less than 3.8 nM. The interaction between PI8 and thrombin 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 approach was used to determine the kinetic constants for the interactions 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 overall 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 an overall K-i of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.