Endogenous cystinyl aminopeptidase in Chinese hamster ovary cells:: characterization by [125I]Ang IV binding and catalytic activity

The angiotensin H C-terminal hexapeptide fragment angiotensin IV (Ang IV) exerts central and cardiovascular effects. Cystinyl ammopeptidase (EC 3.4.11.3), a membrane-associated zinc-dependent metallopepticlase of the M1 family, has recently been found to display high affinity for Ang IV and it was proposed to represent the AT(4) receptor. We present evidence for the presence of endogenous cystinyl aminopeptidase in membranes from Chinese hamster ovary (CHO-K1) cells by binding studies with [I-125]Ang IV and by measuring the cleavage Of L-leucine-p-nitroanilide. The equilibrium dissociation constant of [125 I]Ang IV in saturation binding studies (K-D = 0.90 nM) was similar to the value (K-D = 0.70 nM) calculated from the association and dissociation rates. Binding was displaced with high potency by the "AT(4) receptor" ligands (Ang IV > divalinal(1) -Ang IV similar to LVV-hemorphin-7 similar to LVV-hemorphin-6 > Ang (3-7) > Ang III > Ang (4-8)) but not by AT(1)/AT(2) receptor antagonists. Enzymatic activity in CHO-K1 cell membranes was competitively inhibited upto 94% by Ang Wand other "AT(4) receptor" ligands (Ang IV > Ang III similar to divalinal(1)-Ang IV similar to Ang (3-7) similar to LVV-hemorphin-7 > Ang (4-8) similar to LVV-hemorphin-6). High affinity binding of [I-125]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. (C) 2004 Elsevier Inc. All rights reserved.