Endogenous cystinyl aminopeptidase in Chinese hamster ovary cells: characterization by [125I]Ang IV binding and catalytic activity.
The angiotensin II C-terminal hexapeptide fragment angiotensin IV (Ang IV) exerts central and cardiovascular effects. Cystinyl aminopeptidase (EC 3.4.11.3), a membrane-associated zinc-dependent metallopeptidase of the M1 family, has recently been found to display high affinity for Ang IV and it was proposed to represent the AT4 receptor. We present evidence for the presence of endogenous cystinyl aminopeptidase in membranes from Chinese hamster ovary (CHO-K1) cells by binding studies with [125I]Ang IV and by measuring the cleavage of L-leucine-p-nitroanilide. The equilibrium dissociation constant of [125I]Ang IV in saturation binding studies (KD= 0.90 nM) was similar to the value (KD= 0.70 nM) calculated from the association and dissociation rates. Binding was displaced with high potency by the "AT4 receptor" ligands (Ang IV > divalinal1-Ang IV approximately LVV-hemorphin-7 approximately LVV-hemorphin-6 > Ang (3-7) > Ang III > Ang (4-8)) but not by AT1/AT2 receptor antagonists. Enzymatic activity in CHO-K1 cell membranes was competitively inhibited upto 94% by Ang IV and other "AT4 receptor" ligands (Ang IV > Ang III approximately divalinal1-Ang IV approximately Ang (3-7) approximately LVV-hemorphin-7 > Ang (4-8) approximately LVV-hemorphin-6). High affinity binding of [125I]Ang IV required the presence of metal chelators and the ligands such as Ang IV and LVV-hemorphin-7 displayed higher potency in the binding studies as in the enzyme assay. This difference in potency varied from one peptide to another. These pharmacological properties match those previously reported for the recombinantly-expressed human cystinyl aminopeptidase in embryonal kidney cells.
Demaegdt
Heidi
H
Research Group on Experimental Pharmacology, Department of Molecular and Biochemical Pharmacology, Vrije Universiteit Brussel, Pleinlaan 2, Brussels 1050, Belgium.
Vanderheyden
Patrick
P
De Backer
Jean-Paul
JP
Mosselmans
Sabrina
S
Laeremans
Hilde
H
Le
Minh Tam
MT
Kersemans
Veerle
V
Michotte
Yvette
Y
Vauquelin
Georges
G
D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells / Q000201:enzymology*; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D010122:Cystinyl Aminopeptidase / Q000378:metabolism*; D005260:Female; D007457:Iodine Radioisotopes; D008024:Ligands; D011945:Receptors, Angiotensin / Q000378:metabolism*
C090830:AT4 receptor; D007457:Iodine Radioisotopes; D008024:Ligands; D011945:Receptors, Angiotensin; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; D010122:Cystinyl Aminopeptidase