RESUMO
We investigated the influence of pH and divalent cations (Zn2+, Mg2+ and Ca2+) on high molecular weight kininogen processing by cathepsin B. At pH 6.3, high molecular weight kininogen is hydrolyzed by cathepsin B at three sites generating fragments of 80, 60 and 40 kDa. Cathepsin B has kininogenase activity at this pH which is improved in the absence of divalent cations. At pH 7.35, high molecular weight kininogen is slightly cleaved by cathepsin B into fragments of 60 kDa, and cathepsin B kininogenase activity is impaired. Our results suggest that high molecular weight kininogen is a substrate for cathepsin B under pathophysiological conditions.
Assuntos
Catepsina B/química , Cininogênios/química , Catepsina B/metabolismo , Cátions/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Calicreínas/metabolismo , Cininogênios/efeitos dos fármacos , Cininogênios/metabolismo , Peso Molecular , Especificidade por SubstratoRESUMO
This study was carried out to show the site in kininogen, using synthetic substrates, that is cleaved by a purified human pepsin component with a molecular weight of 35,000 daltons. The study used 4 (four) intramolecularly quenched fluorogenic substrates containing N- and C-terminal sequences around the methionyl-lysyl-bradykinin (MLBK) region of kininogen: Abz-LMKRP-Eddnp, Abz-MISLMKRP-Eddnp, Abz-FRSSR-Eddnp and Abz-RPPGFSPFRSSRQ-Eddnp. The hydrolysis on N-terminal sequences Abz-LMKRP-Eddnp and Abz-MISLMKRP-EDDnp occurred at L-M linkage and on C-terminal sequences Abz-FRSSR-Eddnp, and Abz-RPPGFSPFRSSRQ-Eddnp occurred at S-S linkage. The released peptide Abz-RPPGFSPFRS was able to contract rat uterus muscle. The results suggest that Met-Lys-Bradykinin-Ser, should be the peptide released from human kininogen by a purified human pepsin component.