RESUMO
Low and high molecular weight kininogens (LK and HK), containing 409 and 626 amino acids with masses of ~65 and 120120 kDa after glycosylation, respectively, are coded by a single gene mapped to the human chromosome 3 by alternative splicing of the transcribed mRNA. The NH2-termini Glu(1)-Thr(383) region, identical in LK and HK, contains bradykinin (BK) moieties Arg(363)-Arg(371). LK, HK and their kinin products Lys-BK and BK are involved in several biological processes. They are evolutionarily conserved and only 7 patients, all apparently normal, have been reported to lack them. In one of these patients (Williams'trait), a codon mutation (Arg(178) r stop) has been blamed for the absence of LK and HK. However, using Western blots with 2 monoclonal anti-HK antibodies, one that recognizes the region common to LK and HK and the other that recognizes only HK, I detected ~110-kDa bands in the plasma of this LK/HK-deficient patient vs ~120-kDa bands in normal human and ape plasmas. With polyclonal anti-Lys-BK antibody, which strongly detects BK eleaved at its COOH-terminus in purified HK, I detected ~110-kDa bands in the normal and the deficient plasmas. Western blots with a monoclonal anti-prekallikrein (PK) antibody showed that surface activation of PK and distribution of PK activation products, both dependent on HK, were similar in these plasmas. These findings suggest that a mutant gene yielded a kininogen-like species possibly involving aberrant mRNA splicing - structurally different from normal HK, but apparently with the capacity to carry out seemingly vital HK functions.
Assuntos
Feminino , Humanos , Transtornos da Coagulação Sanguínea/genética , Cininogênio de Alto Peso Molecular/genética , Cininogênio de Baixo Peso Molecular/genética , Cininogênios/genética , Plasma/química , Anticorpos Monoclonais/isolamento & purificação , Transtornos da Coagulação Sanguínea/imunologia , Western Blotting , Cininogênio de Alto Peso Molecular/imunologia , Cininogênio de Baixo Peso Molecular/imunologia , Cininas/isolamento & purificação , Mutação , RNA Mensageiro/genéticaRESUMO
1. Bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg9- bradykinin and des-Arg1- bradykinin were separated by capillary zone electrophoresis in an apparatus constructed in our laboratory which utilizes a novel N2 pulsed laser-induced fluorescence detector. 2. Detection limits of 1.2 fmol for fluorescamine-derivatized bradykinin and 90 attomol for O-phthaldiadehyde-derivatized bradykinin were achieved. 3. This powerful analytical tool is described and its successful application to the measurements of bradykinin after enzymatic release from blood is documented
Assuntos
Animais , Bovinos , Eletroforese , Cininas/isolamento & purificação , Lasers , Bradicinina/isolamento & purificação , Capilares , Fluorescência , Fatores de TempoRESUMO
Partially purified kinin potentiating peptide (KPP) obtained from kininogen-depleted human plasma inhibited lung angiotensin converting enzyme in vitro and potentiated guinea-pig ileum contractions induced by bradykinin (BK), Lys-BK, Met-Lys-BK, Ile-Ser-BK, and Lys-Lys-BK. Contractions evoked by angiotensin II, histamine, acetylcholine, and barium cloride were not potentiated. KPP also potentiated kinin-induced contractions of rat uterus and of guinea-pig ileum pre-incubated with 1,10-phenbabthroline. It in suggested that KPP potentiation is due, at least in part, to a direct effect on kinin receptor(s)