Synergistic modulation of cystinyl aminopeptidase by divalent cation chelators.
Membranes of Chinese hamster ovary (CHO-K1) cells were used to study the opposite modulation of enzyme activity and [125I]Ang IV binding to cystinyl aminopeptidase (EC 3.4.11.3) by divalent cation chelators. Whereas ethylene diamine tetraacetic acid (EDTA) or ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) alone only slightly affected the enzyme activity, 1,10-phenanthrolin (1,10-PHE) produced a complete and concentration-dependent inhibition. Interestingly EDTA (> or =0.05 mM) or EGTA (> or =0.15 mM) enhanced the inhibitory effect of 1,10-PHE. Two-site analysis of the corresponding inhibition curves revealed that EDTA and EGTA converted enzymes with low sensitivity towards 1,10-PHE into enzymes with high sensitivity. The combined inhibition by EDTA (0.1 mM) and 1,10-PHE (0.1 mM) could be prevented and reversed by addition of Zn2+ (at about 0.04-0.1 mM). In contrast, specific binding of [125I]Ang IV was enhanced in the presence of 1,10-PHE. Binding was only slightly affected by EDTA or EGTA alone. Furthermore, the stimulatory effect of 1,10-PHE was potentiated by EDTA (> or =0.05 mM) as well as EGTA (> or =0.15 mM). In the presence of EDTA (0.1 mM) and 1,10-PHE (0.1 mM), specific [125I]Ang IV binding was completely inhibited by Zn2+ (IC50= 39.7 +/- 6.2 microM). The present data show that divalent cations such as Zn2+ are essential for the enzyme activity of cystinyl aminopeptidase and inhibitory for [125I]Ang IV binding. Modulation of the effects of 1,10-PHE by other chelators such as EDTA or EGTA, suggests that, in addition to the binding site for zinc in the catalytic site, cystinyl aminopeptidase also bears a regulatory divalent cation binding site.
Research Group on Experimental Pharmacology, Department of Molecular and Biochemical Pharmacology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
Heidi
H
Demaegdt
Hilde
H
Laeremans
Jean-Paul
JP
De Backer
Sabrina
S
Mosselmans
Minh Tam
MT
Le
Veerle
V
Kersemans
Yvette
Y
Michotte
Georges
G
Vauquelin
Patrick M L
PM
Vanderheyden
D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002413:Cations, Divalent / Q000494:pharmacology*; D002614:Chelating Agents; D006224:Cricetinae; D010122:Cystinyl Aminopeptidase / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D007457:Iodine Radioisotopes; D010618:Phenanthrolines / Q000494:pharmacology; D011869:Radioligand Assay
D002413:Cations, Divalent; D002614:Chelating Agents; D007457:Iodine Radioisotopes; D010618:Phenanthrolines; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; D004533:Egtazic Acid; D004492:Edetic Acid; D010122:Cystinyl Aminopeptidase