Angiotensin converting enzymes from human urine of mild hypertensive untreated patients resemble the N-terminal fragment of human angiotensin I-converting enzyme.
Angiotensin I-converting enzyme (ACE) activity was analyzed in human urine collected from mild hypertensive untreated patients. DEAE-cellulose chromatography using linear gradient elution revealed two forms of angiotensin I-converting enzyme, eluted in the conductivity of 0.75 and 1.25 mS. The fractions of each conductivity were pooled and submitted to direct gel filtration in an AcA-34 column, and the apparent molecular weights of urinary ACEs were estimated as 90 kDa (for ACE eluted in 0.75 mS) and 65 kDa (for ACE eluted in 1.25 mS). Both enzymes have a K(i) of the order of 10(-7) M for the specific inhibitors studied, and are able to hydrolyze luteinizing hormone-releasing hormone and N-acetyl-Ser-Asp-Lys-Pro as described for N-domain ACE. By Western blot analysis, both peaks were recognized by ACE-specific antibody Y4, confirming the molecular weight already described. A plate precipitation assay using monoclonal antibodies to the N-domain of ACE showed that both forms of ACE binds with all monoclonal antibodies to the active N-domain ACE, suggesting that these forms of human urine ACEs resemble the N-fragment of ACE. The HP2 ACE (65 kDa) is similar to low molecular weight (LMW) ACE from normal subjects, and the HP2 ACE (90 kDa) is different from high molecular weight (190 kDa) and LMW (65 kDa) normal ACEs. The 90 kDa ACE could have an important role in development of hypertension. It will be fundamental to elucidate the molecular mechanism responsible for the genesis of this isoform.
Casarini
D E
DE
Universidade Federal de São Paulo, Escola Paulista de Medicina, Departamento de Medicina, Disciplina de Nefrologia, Rua Botucatu, 740, CEP 04023-900, SP, São Paulo, Brazil. dulce@nefro.epm.br
Plavinik
F L
FL
Zanella
M T
MT
Marson
O
O
Krieger
J E
JE
Hirata
I Y
IY
Stella
R C
RC
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D006973:Hypertension / Q000378:metabolism* / Q000652:urine*; D007700:Kinetics; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000652:urine*; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D020033:Protein Isoforms; D007703:Peptidyl-Dipeptidase A