Relationship between the cysteine-proteinase-inhibitory function of rat T kininogen and the release of immunoreactive kinin upon trypsin treatment

Abstract

The potential kininogenic function of rat T kininogen has been studied in parallel with the cysteine-proteinaseinhibitory function also carried by this molecule. Proteolytic cleavage of the molecule was observed upon incubation with catalytic amounts of trypsin. These conditions do not permit any significant release of immunoreactive kinin and do not modify the total papain-inhibiting capacity of T kininogen. As trypsin concentration increases in the reaction mixture, immunoreactive kinin is liberated and the total papain-inhibiting capacity decreases accordingly, as indicated by titration studies. This decrease, however, does not exceed 50% of the initial value even at a trypsin concentration as high as 75 pM, indicating that only one of the two inhibitory sites has been inactivated. The remaining inhibitory fragment corresponds to a peptide of apparent M , 24000, which binds papain at least as well as native T kininogen.T kininogen, therefore, appears as a potent proteinase inhibitor and/or a proteinase inhibitor precursor, whereas its kininogenic function remains questionable since no specific kininogenase able to release T kinin or another kinin under physiologically compatible conditions has been found so far. In addition to its potential function as kinin precursor, T kininogen as well as other kininogens is a potent cysteine proteinase inhibitor identical with the previously reported u1 cysteine proteinase inhibitor [lo]. At variance with other kininogens, however, T kininogen also behaves as a typical acute-phase reactant since its concentration may be increased by 5-10 during an inflammatory state [lo].

Purification and characterization of a new cystatin inhibitor from Taiwan cobra (Naja naja atra) venom

“…Cobra cystatin was titrated with papain, previously titrated with the irreversible epoxide inhibitor 1-trans-epoxysuccinylleucylamido-(4-guanidino)butane (E64) as described in [19]. A final papain concentration of 30 nM in 0.1 M phosphate buffer (pH 6.8)\1 mM EDTA\2 mM dithiothreitol\0.1 % Brij35, to which various volumes of cystatin were added, was used.…”

“…Apparent inhibition constants (K i,app ) were determined as previously described [19,20] under experimental conditions that gave a non-linear dose-response curve. Z-Phe-Arg-MCA (3 µM final concentration) was used as the fluorigenic substrate for all four enzymes and the assay buffer (pH 6.8 for papain and pH 6.0 for cathepsins B, L and S) was 0.1 M phosphate buffer\1 mM EDTA\2 mM dithiothreitol\0.1 % Brij35.…”

The Occluding Loop of Cathepsin B Prevents Its Effective Inhibition by Human Kininogens

“…73 Inhibition constants K i(app) were determined under experimental conditions that gave nonlinear dose-response curves as described elsewhere. 74 Data were plotted using the Easson and Stedman procedure as described by Bieth 75 and fitted with Enzfitter software (Biosoft, Cambridge). Enzyme concentration [E 0 ] was at least 1-to 10-fold higher than the K i value, and less than 5% of substrate (Z-Phe-Arg-AMC) was hydrolyzed during these experiments.…”

Immunological characterization of rat kininogens with monoclonal antibodies to T‐kininogen.

“…The mAb TK [16][17][18][19][20][21][22][23][24][25][26][27][28].5b.2, tested by ELISA, did not crossreact with rat H-kininogen or bovine kininogen (up to 100 pg/ i d ) or with mouse or human plasma. This antibody recognized the 20-kDa peptide; when increasing amounts of the 20-kDa peptide (0.03-3 pg/ml) were incubated with TK 16-28.5b.2, a curve parallel to that of native T-kininogen was obtained (not shown).…”

“…This also suggests that these antibodies do not cross-react with L-kininogen, which has the same heavy chain as H-kininogen [38]. For the same reason, the two antibodies TK [17][18][19][20][21][22][23][24][25][26][27].a and TK 20-4.1 that recognize the H-kininogen heavy chain probably cross-react with the L-kininogen. Antibodies specific to each of the three T-kininogen domains were identified by comparing their specificity toward different proteolytic fragments.…”