Metal ion modulation of cystinyl aminopeptidase.
Cystinyl aminopeptidase has one Zn2+-binding motif and is a member of the M1 aminopeptidase family. Ion modulation of its catalytic activity was studied in membranes of CHO-K1 cells (Chinese-hamster ovary K1 cells) using L-leucine-p-nitroanilide as substrate. The planar bidentate chelators 1,10-phenanthroline and 2,2'-bipyridine inhibited the activity in a concentration-dependent manner with Hill slopes of 3.32+/-1.78 and 2.10+/-0.26 respectively. The acetic acid-containing chelators EDTA, EGTA and DTPA (diethylenetriamine-N,N,N',N'',N''-penta-acetic acid) weakly affected the activity, but they increased the potency of the planar chelators up to a limit, at which Hill slopes became close to unity. Moreover, competition between 1,10-phenanthroline and the substrate only took place in the presence of EDTA. These findings are compatible with a model in which the bidentate chelators inhibit enzyme activity by decreasing the free Zn2+ concentration. By removing a modulatory ion from an allosteric site at the enzyme, the acetic acid-containing chelators facilitate the direct interaction between the bidentate chelators and the catalytic Zn2+. The inhibitory effect of EDTA plus 1,10-phenanthroline could be completely reversed by Zn2+. Ca2+ and Mg2+ increased the potency of Zn2+ for this process. This is expected if they interact with the modulatory site to decrease the sensitivity of the enzyme towards 1,10-phenanthroline. Conversely, the bidendate chelators increased the high-affinity [125I]angiotensin IV binding to the membranes and this was potentiated by the acetic acid-containing chelators. These findings support the concept that high-affinity [125I]angiotensin IV binding, previously referred to as 'AT4 receptor binding', only occurs for the cystinyl aminopeptidase apoenzyme.
Laeremans
Hilde
H
Department of Molecular and Biochemical Pharmacology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
Demaegdt
Heidi
H
De Backer
Jean-Paul
JP
Le
Minh T
MT
Kersemans
Veerle
V
Michotte
Yvette
Y
Vauquelin
Georges
G
Vanderheyden
Patrick M L
PM
D015082:2,2'-Dipyridyl / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry*; D002413:Cations, Divalent / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D006224:Cricetinae; D010122:Cystinyl Aminopeptidase / Q000737:chemistry* / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry; D004533:Egtazic Acid / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry*; D008670:Metals / Q000737:chemistry*; D004369:Pentetic Acid / Q000737:chemistry; D010618:Phenanthrolines / Q000737:chemistry; D011485:Protein Binding; D015032:Zinc / Q000737:chemistry*
D002413:Cations, Divalent; D002614:Chelating Agents; D008670:Metals; D010618:Phenanthrolines; D004533:Egtazic Acid; D015082:2,2'-Dipyridyl; D004369:Pentetic Acid; D004492:Edetic Acid; D010122:Cystinyl Aminopeptidase; D008274:Magnesium; D015032:Zinc; D002118:Calcium; C025205:1,10-phenanthroline