Influence of CTI on rPRCP activation of PK
. | Chromogenic substrate hydrolysis . | . | |
|---|---|---|---|
| Reactants . | H-D-Pro-Phe-Arg-pNA OD/h ± SD . | H-Gly-Pro-pNA OD/h ± SD . | |
| HK + PK* | 0.09 ± 0.03 | 0.11 ± 0.02 | |
| HK + PK + rPRCP* | 0.54 ± 0.03 | 0.54 ± 0.01 | |
| HK + PK + rPRCP + CTI*‡ | 0 ± 0.14 | 0.49 ± 0.02 | |
| Soluble kallikrein† | 0.82 ± 0.08 | 0.12 ± 0.01 | |
| Soluble rPRCP† | 0.08 ± 0.05 | 0.59 ± 0.08 | |
| Soluble kallikrein + CTI†‡ | 0.75 ± 0.04 | — | |
| Soluble rPRCP + CTI†‡ | — | 0.52 ± 0.03 | |
. | Chromogenic substrate hydrolysis . | . | |
|---|---|---|---|
| Reactants . | H-D-Pro-Phe-Arg-pNA OD/h ± SD . | H-Gly-Pro-pNA OD/h ± SD . | |
| HK + PK* | 0.09 ± 0.03 | 0.11 ± 0.02 | |
| HK + PK + rPRCP* | 0.54 ± 0.03 | 0.54 ± 0.01 | |
| HK + PK + rPRCP + CTI*‡ | 0 ± 0.14 | 0.49 ± 0.02 | |
| Soluble kallikrein† | 0.82 ± 0.08 | 0.12 ± 0.01 | |
| Soluble rPRCP† | 0.08 ± 0.05 | 0.59 ± 0.08 | |
| Soluble kallikrein + CTI†‡ | 0.75 ± 0.04 | — | |
| Soluble rPRCP + CTI†‡ | — | 0.52 ± 0.03 | |
Each reactant was added to a microtiter plate cuvette well at the indicated concentration. When plasma kallikrein hydrolytic activity was measured, the reactants were in HEPES carbonate buffer, pH 7.4, and 0.8 mM H-D-Pro-Phe-Arg-pNA was added. When rPRCP activity was measured by the hydrolysis of 3 mM H-Gly-Pro-pNA, the reaction was in 0.2 M sodium acetate, 0.15 KCI, pH 5.5 buffer.—indicates no data.
In the reactions HK + PK in the absence or presence of additions, the HK was linked to microtiter plate wells at 2μg/mL in sodium carbonate buffer, pH 9.6, by overnight incubation at 4°C. Afterward the plate was washed with HEPES carbonate buffer, pH 7.4, containing 0.1% gelatin or 0.2 M sodium acetate, 0.15 M KCI, pH 5.5, containing 0.1% gelatin followed by the addition of 20 nM PK in the absence or presence of 10 to 20 nM rPRCP in the absence or presence of 1μM CTI. After incubation, the wells were washed with the appropriate buffer for the enzymatic reaction. In experiments measuring plasma kallikrein, 0.8 mM H-D-Pro-Phe-Arg-pNA in HEPES carbonate buffer, pH 7.4, was used; in other experiments measuring rPRCP, 3 mM H-Gly-Pro-pNA in 0.2 M sodium acetate, 0.15 M KCI, pH 5.5, was used. In all instances, the data provided are the mean ± SD of 3 or more individual experiments.
Enzyme hydrolysis of plasma kallikrein (0.3μM), rPRCP, rPRCP + CTI, or plasma kallikrein + CTI with each respective substrate was performed in solution, as indicated by the prefix “soluble.”
CTI added at 1μM final concentration to the reaction.