title: Paramagnetic bradykinin analogues as substrates for angiotensin I-converting enzyme: Pharmacological and conformation studies.
abstract: This study uses EPR, CD, and fluorescence spectroscopy to examine the structure of bradykinin (BK) analogues attaching the paramagnetic amino acid-type Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 3, 7, and 9. The data were correlated with the potencies in muscle contractile experiments and the substrate properties towards the angiotensin I-converting enzyme (ACE). A study of the biological activities in guinea pig ileum and rat uterus indicated that only Toac0-BK partially maintained its native biological potency among the tested peptides. This and its counterpart, Toac3-BK, maintained the ability to act as ACE substrates. These results indicate that peptides bearing Toac probe far from the ACE cleavage sites were more susceptible to hydrolysis by ACE. The results also emphasize the existence of a finer control for BK-receptor interaction than for BK binding at the catalytic site of this metallodipetidase. The kinetic kcat/Km values decreased from 202.7 to 38.9μM-1min-1for BK and Toac3-BK, respectively. EPR, CD, and fluorescence experiments reveal a direct relationship between the structure and activity of these paramagnetic peptides. In contrast to the turn-folded structures of the Toac-internally labeled peptides, more extended conformations were displayed by N- or C-terminally Toac-labeled analogues. Lastly, this work supports the feasibility of monitoring the progress of the ACE-hydrolytic process of Toac-attached peptides by examining time-dependent EPR spectral variations.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP, Brazil.
authors.forename: Luis Gustavo Deus
authors.initials: LG
authors.lastname: Teixeira
authors.affiliation: Department of Physiological Sciences, Santa Casa de Sao Paulo, School of Medical Sciences, 01221-020 Sao Paulo, SP, Brazil.
authors.forename: Luciana
authors.initials: L
authors.lastname: Malavolta
authors.affiliation: Department of Health Informatics, Universidade Federal de Sao Paulo, 04023-062 Sao Paulo, SP, Brazil.
authors.forename: Patrícia Alessandra
authors.initials: PA
authors.lastname: Bersanetti
authors.affiliation: Department of Biochemistry, Institute of Chemistry, Universidade de Sao Paulo, 05513-970 Sao Paulo, SP, Brazil.
authors.forename: Shirley
authors.initials: S
authors.lastname: Schreier
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP, Brazil.
authors.forename: Adriana K
authors.initials: AK
authors.lastname: Carmona
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP, Brazil. Electronic address: cnakaie@unifesp.br.
authors.forename: Clovis R
authors.initials: CR
authors.lastname: Nakaie
mesh_terms: D000806:Angiotensin-Converting Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D001920:Bradykinin / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D006168:Guinea Pigs; D007082:Ileum / Q000187:drug effects* / Q000378:metabolism; D008968:Molecular Conformation; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship; D014599:Uterus / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D007703:Peptidyl-Dipeptidase A; D001920:Bradykinin
keywords: ACE; Angiotensin I-converting enzyme; Biological activity; Bradykinin; Spin label; Toac