Inhibitory effect of reactive oxygen species on angiotensin I-converting enzyme (kininase II).
1. Somatic angiotensin I-converting enzyme (ACE) is a protein that contains two similar domains (N- and C-terminal), each possessing an active site. We have examined the effects of a generator of hydroxyl radicals (g*OH: 2,2'-azo-bis(2-amidinopropane)) and hydrogen peroxide (H2O2) on ACE using an in vitro approach. 2. The generator of hydroxyl radicals inactivated ACE in a time (2-6 h)- and concentration (0.3-3 mmol/L)-dependent manner at 37 degrees C. When ACE was coincubated for 4 h with g*OH (3 mmol/L), its activity decreased by 70%. Addition of dimethylthiourea or mannitol + methionine, two *OH scavengers, resulted in a significant protection of ACE activity. Mercaptoethanol and dithiotreitol, two thiol-reducing agents, also efficiently protected ACE activity. 3. The hydrolysis of two natural and domain-specific substrates was explored. The hydrolysis of angiotensin I, preferentially cleaved by the C-domain, was significantly inhibited (57-58%) after 4 h exposure to g*OH (0.3-1 mmol/L). Under the same conditions of exposure, the hydrolysis of N-acetyl-Ser-Asp-Lys-Pro, a specific substrate for the N-domain, was only slightly inhibited by 1 mmol/L g*OH. 4. Hydrogen peroxide, another source of *OH, was used. After exposure to H2O2 (3 mmol/L; 4 h), an 89% decrease in ACE activity was observed. Pretreatment with the iron chelator deferoxamine (1 mmol/L) attenuated H2O2-mediated ACE inactivation, demonstrating that the effect of H2O2 was partly due to its conversion into *OH (Fenton reaction). 5. In summary, our findings demonstrate that g*OH and H2O2 inhibit ACE activity and suggest a preferential action of g*OH on the C-domain of the enzyme.
Michel
B
B
Institut de Pharmacologie, Université Louis Pasteur de Strasbourg, France. Bruno.Michel@chru-strasbourg.fr
Grima
M
M
Nirina
L B
LB
Ingert
C
C
Coquard
C
C
Barthelmebs
M
M
Imbs
J L
JL
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000737:chemistry*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D016166:Free Radical Scavengers / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D017665:Hydroxyl Radical; D007202:Indicators and Reagents; D007700:Kinetics; D016877:Oxidants / Q000737:chemistry; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000302:isolation & purification; D017382:Reactive Oxygen Species / Q000502:physiology*; D019163:Reducing Agents / Q000737:chemistry; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013552:Swine
D000806:Angiotensin-Converting Enzyme Inhibitors; D016166:Free Radical Scavengers; D007202:Indicators and Reagents; D016877:Oxidants; D017382:Reactive Oxygen Species; D019163:Reducing Agents; D013439:Sulfhydryl Reagents; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D007703:Peptidyl-Dipeptidase A