Angiotensin converting-like enzymes from urine of untreated renovascular hypertensive and normal patients: purification and characterization.
Angiotensin converting-like enzymes (ACE) were isolated from urine of normal (P0N, P1N and P2N) and untreated renovascular hypertensive (P0, P1 and P2) patients. The urine were submitted to ion exchange chromatography. Enzymes P0 and P0N were eluted with the equilibrium buffer (0.02 M Tris-HCl, pH 7.0), while P1, P1N, P2 and P2N with ionic strength linear gradient of 0.02-0.5 M Tris-HCl, pH 7.0 in 0.7 mS and P2 and P2N in 1.2 mS conductance. The active fractions were submitted to gel filtration in Sephadex G-150, equilibrated and performed with 0.05 M Tris-HCl/0.15 M NaCl buffer, pH 8.0. All enzymes were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (molecular mass: P0, P2 and P2N about 60 kDa; P1, 95 kDa and P21N 170 kDa). The enzymes were recognized by Y1 polyclonal antibody raised against human renal ACE. The K(M) values were in millimolar order for hippuryl-L-His-Leu (HHL) while for benzyloxycarbonyl-Phe-L-His-Leu (ZFHL) they were in 10(-4) M order. The enzymes were able to hydrolyze angiotensin I (AI) (P0 and P0N about 25%, P1 and P1N about 70%, P2 100% and P2N 66%) and bradykinin (BK) (P0N 22%, P1N 81%, P2N 62%, P0 and P1 50% and P2 35%), and their activities were inhibited by captopril.
Costa
R H
RH
Department of Biochemistry, Universidade Federal de São Paulo-Escola Paulista de Medicina, SP, Brazil.
Casarini
D E
DE
Plavnik
F L
FL
Marson
O
O
Alves
K B
KB
D000328:Adult; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D006978:Hypertension, Renovascular / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D007703:Peptidyl-Dipeptidase A / Q000302:isolation & purification / Q000652:urine*
D000806:Angiotensin-Converting Enzyme Inhibitors; D007703:Peptidyl-Dipeptidase A