Characterisaton of human plasma thiol proteinase inhibitors.

Earlier, we had reported purification of three thiol proteinase inhibitors (TPI-1 of 70 kDa, TPI-3 of 195 kDa and TPI-4 of 497 kDa) from human plasma. In the present study we report that TPI-1 binds to papain in the stoichiometry ratio (E/I) of 1:1 while TPI-3 and TPI-4 bind in the ratio of 1.5:1 and 3.2:1 respectively. The K(m) for papain with BAPNA as substrate and Kcat/K(m) values for TPI-1, TPI-3 and TPI-4 were 2.7 x 10(-6) M, 0.84 nM/sec; 3.2 x 10(-6) M, 0.75 nM/sec; and 3.6 x 10(-6) M, 0.72 nM/sec respectively. The Ki values were found to be 1.48 nM for TPI-1, 0.133 nM for TPI-3 and 0.117 nM for TPI-4. The UV absorption and fluorescence emission spectra study suggest involvement of aromatic residues in the binding process. This study suggests that TPI-4 is the most potent inhibitor of thiol proteinases.

Purification and physicochemical studies of human plasma thiol proteinase inhibitors.

The physicochemical properties of thiol proteinase inhibitors (TPI) isolated from outdated human blood have been studied. A simple technique which includes ammonium sulphate precipitation, Sephadex G-200 gel filtration and ion-exchange chromatography led to the isolation of 4 isolates namely TPI-1, TPI-2, TPI-3 and TPI-4 having molecular mass of 70, 155, 195 and 497 kDa respectively. The latter two forms are the new isolates unreported previously. They exhibit similar pH stability, inhibition spectra with papain, cathepsin B and trypsin, antigenic properties and glycoprotein nature. The TPI-4, however, was found to be most heat stable showing no decrease in inhibitory activity when heated upto 70 degrees C for 30 min. Our work suggests that TPI-3 and TPI-4 are the oligomers of TPI-1.

Kinins mediate the inhibition of atrial natriuretic peptide diuretic effect induced by pepsanurin

Pepsanurin is a peptidic fraction resulting from pepsin digestion of plasma globulins, that inhibits ANP renal excretory actions. We studied whether kinin-like peptides mediate the anti-ANP effect by testing if pepsanurin: 1) was blocked by the kinin B12 receptor antagonist HOE-140, 2) was produced from kininogen, and 3) was mimicked by bradykinin. Anti-ANP activity was assessed in anesthetized female rats by comparing the excretory response to two ANP boluses (0.5 mug iv) given before and after ip injection of test samples. Pepsanurin from human or rat plasma (1-5 mL/Kg), and bradykinin (5-20 mug/Kg), dose-relatedly inhibited ANP-induced water, sodium, potassium and cyclic GMP urinary excretion, without affecting arterial blood pressure. The same effect was exerted by pepsin hydrolysates of purified kininogen, whereas hydrolysates of kininogen-free plasma had no effect. HOE-140 (5 mug, iv) did not alter baseline, or ANP-induced excretion, but blocked the anti-ANP effects of pepsanurin. Histamine (15 mug/Kg) plus seroalbumin hydrolysates did not affect ANP response, despite inducing larger peritoneal fluid accumulation as compared with pepsanurin or bradykinin. We concluded that kinins cleaved from kininogen mediate the anti-ANP effects of pepsanurin by activation of kinin B2 receptors, independently of changes in systemic arterial pressure or peritoneal fluid sequestration.