Rat tissue kallikrein releases a kallidin-like peptide from rat low-molecular-weight kininogen.

The kallikrein-kinin system is subdivided into the plasma and tissue-kallikrein-kinin system, with bradykinin (BK) and kallidin (KAL) (Lys(0)-bradykinin) as functional peptides. This occurs in both humans and other mammals. Both peptides are released by plasma and tissue-kallikrein. BK, but not KAL, has been detected in rats until now. One can explain this observation by the structural differences found in the sequence of rat high- and low-molecular kininogen containing an Arg-residue instead of a Lys-residue in front of the N-terminus of the BK sequence. Nevertheless, we were able to measure a kallidin-like peptide (KLP), in rat plasma and urine, using a specific KAL antiserum. In order to confirm our data, we isolated low-molecular-weight kininogen from rat plasma and incubated it with purified rat glandular kallikrein. The generated peptide was retained on a high-pressure liquid chromatography column and displaced by an excess of angiotensin I. The KLP-containing fraction was identified with the KLP radioimmunoassay. A specific ion signal with a mass to charge ratio (m/z) of 1216.73 was detected with matrix-assisted laser desorption/ionization mass spectrometry. As proposed earlier, the structure of this peptide is Arg(1)-KAL, instead of Lys(1)-KAL. The structural similarity between the Lys- and the Arg-residue explains the high crossreactivity (80%) of KLP with the specific KAL antibody. The incubation of KLP with angiotensin-converting enzyme yields two molecules with masses of 913.4 and 729.3 containing the sequence H-Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-OH and H-Arg-Arg-Pro-Pro-Gly-Phe-OH. The enzymatic cleavage could be inhibited by captopril. The data suggest that in rats, as in other mammals, the tissue kallikrein-kinin system mediates its physiological effects via a kallidin-like peptide, which is Arg(1)-kallidin (Arg(0)-bradykinin).

Prediction of band profiles of mixtures of bradykinin and kallidin from data acquired by competitive frontal analysis.

The competitive adsorption isotherms of two closely related peptides, bradykinin and kallidin, were measured by frontal analysis on a Zorbax SB-C18 microbore column. An aqueous soluton at 20% acetonitrile (0.1% TFA) was used as the mobile phase. The competitive isotherm data were fitted to four different models: Langmuir, Bilangmuir, Langmuir-Freundlich, and Toth. These data fitted best to a Bilangmuir isotherm model. The influence of the pressure on the retention factors of the two peptides was found to be small and was not investigated in detail. The band profiles of large samples of the single components and of their mixtures were recorded. The overloaded profiles calculated using either the equilibrium-dispersive or POR model are in excellent agreement with the experimental profiles in all cases. Our results confirm that the competitive isotherm data derived from mixtures may suffice for a reasonably accurate prediction of the band profiles of all mixtures of the two components, provided their composition is close to 1/1.

Electrophoresis separation in open microchannels. A method for coupling electrophoresis with MALDI-MS.

The separation of biological mixtures in open micro-channels using electrophoresis with rapid and simple coupling to mass spectrometry is introduced. Rapid open-access channel electrophoresis employs microchannels that are manufactured on microchips. Separation is performed in the open channels, and the chips are transferred to a matrix-assisted laser desorption/ionization (MALDI) source after the solvent is evaporated. The matrix (2,5-dihydroxybenzoic acid) is placed in the solution with the run buffer before the separation of the analyte components. After separation, the solvent is evaporated and the microchip is ready for MALDI-MS analysis. The microchip is placed directly into a specially designed ion source of an external source Fourier transform mass spectrometry instrument. Separation of simple mixtures containing oligosaccharides and peptides is shown.

Characterization of a kallidin receptor in the eel intestine.

Edman degradation of an eel bradykinin (BK) -like peptide isolated and detected by gel filtration and HPLC and RIA gave an amino acid sequence of Arg1-Pro-Pro-Gly-X-Ser-Pro-Leu-Arg9. Kallidin but not BK and des-Arg9-BK contracted eel intestine. The contractile effect of kallidin was not decreased by B1 and B2 receptor antagonists (up to 10(-6)M), nor by anticholinergics, antiadrenergics, ganglion blockers and an angiotensin II receptor antagonist but was attenuated by 10(-5)M indomethacin. Kallidin appears to interact with a receptor different from the BK B1 and B2 receptor types and prostaglandins may participate in the response.

Isolation of [hydroxyproline3]Lysyl-bradykinin formed by kallikrein from human plasma protein.

[Hydroxyproline3]Lysyl-bradykinin ([Hyp3]Lys-BK), a new kinin was isolated, besides Lysyl-bradykinin (Lys-BK), from the reaction mixture of human plasma protein Cohn's fraction IV-4 with hog pancreatic kallikrein. The liberated kinins were isolated by procedures including ethanol extraction, Sephadex G-15, CM cellulose and reverse-phase high performance liquid chromatography and quantitated by radioimmunoassay. On HPLC, two peaks of immunoreactive kinins emerged. Peak 1, an unknown kinin proceeded to peak 2 which had an identical retention time to that of Lys-BK. The amino acid sequence of the unknown peak 1 proved to be Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg, or [Hydroxyproline3]Lys-BK, and peak 2 Lys-BK. The ratio of the amounts of two kinins thus formed were [Hyp3]Lys-BK 25 +/- 4% and Lys-BK 75 +/- 4%. The existence of [Hyp3]Lys-BK suggests a presence of a new kininogen containing [Hyp3]Lys-BK in human plasma protein.

Quantification, isolation and structural determination of bradykinin and hydroxyprolyl-bradykinin in tumor ascites.

The presence of kinins in ascitic tumor fluids from rodents and human patients was identified and quantified. In bioassay, kinin content was found to be 1 to 40 ng/ml, and by enzyme immunoassay, 0.6 to 2.5 ng/ml. In particular, a high kinin content, 40 ng/ml, was found in the ascites of a gastric cancer patient by bioassay. Purification of this kinin in the ascites from the gastric cancer patient was performed by ethanol precipitation, gel filtration and reversed-phase high-performance liquid chromatography (HPLC). Two peaks (peak A and peak B) showed kinin activity. Peak A did not correspond to either bradykinin or other known kinins, such as lysyl-bradykinin and T-kinin, whereas peak B corresponded to bradykinin. Peak A contained 8 amino acid residues from bradykinin minus one proline plus an additional hydroxyproline. Sequence analysis of peak A showed that the proline at the third amino acid residue of bradykinin was replaced by hydroxyproline. The retention time of peak A on reversed-phase HPLC was exactly the same as that of synthetic hydroxyprolyl3-bradykinin (Hyp3-bradykinin) but was distinguishable from des-Pro3-bradykinin. Thus, these results demonstrate for the first time the presence of Hyp3-bradykinin in mammalian system.

Determination of bradykinin, kallidin and Met-Lys-bradykinin by high performance liquid chromatography.

A high performance liquid chromatography system was developed for the quantitative separation and determination of bradykinin, kallidin and Met-Lys-bradykinin using a reversed-phase column (Nucleosil 5 C8) and an isocratic buffer system. The lower limit of detection is 20 pmol for bradykinin. The intra-variation coefficient of the assay is between 1.8 and 5.7%. The recovery of bradykinin is higher than 96%. Addition of human serum albumin to the assay system in a concentration of 40% lead to a remarkable reduction of the detectable kinin level, which indicates an adsorption of kinins to serum albumin.