Purification and characterization of endopeptidase H2, a kinin inactivating serine proteinase (kininase) from human urine.
1. A kinin-inactivating chymotrypsin-like serine-endopeptidase was purified 202-fold from human urine by DEAE-cellulose chromatography, gel filtration, DEAE/HPLC chromatography and affinity chromatography. It hydrolyzed bradykinin at the Phe5-Ser6 peptide bond at a rate of 1.090 mumol min-1 mg protein-1 at pH 8.0 and 37 degrees C. The molecular weight of this endopeptidase H2, estimated by SDS-polyacrylamide gel electrophoresis and by gel filtration, was 60 kDa, and its optimum pH for bradykinin hydrolysis was near 8.5. 2. Bradykinin inactivating activity was inhibited 100% by the serine-proteinase inhibitor PMFS (1 mM) and the chymotrypsin inhibitor TPCK (5 mM). Reagents such as 2-mercaptoethanol (3 mM) and pOH-mercuribenzoate (3 mM) inhibited the enzyme by 100% and 67%, respectively. 3. Endopeptidase H2 hydrolyzes the Phe-Ser bond of peptides related to bradykinin and its activity appears to be limited to peptide chains of < or = 18 amino acid residues since it does not hydrolyze BAM 22, peptide E or kininogen. 4. The molecular size and inhibition profile suggested that endopeptidase H2 differs from the serine-proteinases previously described in rat liver, rat hepatic endothelium, rat and rabbit brain. 5. The physiological role of endopeptidase H2 may be a link between the kinin and neuropeptide systems in the control of water-electrolyte balance.
Casarini
D E
DE
Departamento de Medicina, Escola Paulista de Medicina, São Paulo, Brasil.
Stella
R C
RC
Araújo
M S
MS
Sampaio
C A
CA
D000818:Animals; D001920:Bradykinin / Q000378:metabolism; D002853:Chromatography, Liquid; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007705:Kinins / Q000037:antagonists & inhibitors; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000187:drug effects / Q000302:isolation & purification* / Q000652:urine; D013997:Time Factors; D014882:Water-Electrolyte Balance
D007705:Kinins; D012697:Serine Endopeptidases; D001920:Bradykinin