RESUMO
It has been proposed that a cysteine proteinase inhibitor (CPI) found in the ascitic fluid of Sarcoma 180 tumor-bearing mice is a kind of kininogen (Itoh, N., Yokota, S., Takagishi, U., Hatta, A., and Okamaoto, H. (1987) Cancer Res. 47, 5560-5565). The first 40 NH2-terminal residues and 54 residues of the COOH-terminal sequence, including the bradykinin moiety of highly purified ascites CPI, were determined and compared with those of mammalian low molecular weight kininogens (LMWK). The significant identity between these amino acid sequences with those of other mammalian LMWKs suggests that ascites CPI corresponds precisely to mouse LMWK. This kininogen has a light chain composed of 43 amino acid residues, which contains a unique Met-Ala-Arg-bradykinin sequence. Hydroxyproline, which was recently identified in the bradykinin sequence of kininogen from the ascitic fluid of a cancer patient, was not found in the kinin moiety of this mouse kininogen. Among purified glandular kallikreins from human, hog, rat, and mouse, only mouse submaxillary gland kallikrein was able to release bradykinin from this kininogen. Kinetic studies using a newly synthesized fluorogenic substrate, N-t-butoxycarbonyl-Met-Ala-Arg-MCA, revealed that mouse kallikrein hydrolyzes this substrate approximately 80-fold faster than does hog kallikrein, suggesting that the unique Met-Ala-Arg-bradykinin sequence is responsible for the varied susceptibility of mouse kininogen to different kallikreins.
Assuntos
Inibidores de Cisteína Proteinase/isolamento & purificação , Calicreínas/metabolismo , Cininogênios/isolamento & purificação , Sarcoma 180/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Ascite , Carboidratos/análise , Bovinos , Humanos , Calicreínas/isolamento & purificação , Cininogênios/genética , Camundongos , Dados de Sequência Molecular , Peso Molecular , Ratos , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato , SuínosAssuntos
Cininogênios/análise , Neoplasias Pulmonares/fisiopatologia , Sarcoma Experimental/fisiopatologia , Animais , Ascite/fisiopatologia , Divisão Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/sangue , Inibidores de Cisteína Proteinase/farmacologia , Replicação do DNA/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Valores de Referência , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Studies were carried out in order to characterize the kininogen in rat urine. Rat urine contained a component which was cross-reactive with antibody to rat plasma T-kininogen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rat urine revealed a single antigenic band corresponding to the molecular weight of plasma T-kininogen. Induction of acute inflammation in rats by an injection of lipopolysaccharide caused an increase in the urinary excretion of immunoreactive T-kininogen in parallel with an elevation of plasma T-kininogen. Kininogen partially purified from rat urine by affinity chromatography using S-carboxymethylated papain-agarose liberated only T-kinin upon trypsinization, but not upon treatment with rat glandular kallikreins. From these results, we conclude that T-kininogen is the major kininogen present in rat urine.