Co-solubilization of bradykinin B2 receptors and angiotensin-converting enzyme from guinea pig lung membranes.
Bradykinin B2 receptor-like binding activity was solubilized from guinea pig lung using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (Chaps). The binding of [3H]bradykinin to the soluble fraction was time-dependent and saturable. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of binding sites with a Kd of 696 pM and a Bmax of 57 fmol/mg protein. Unlabelled bradykinin and B2 antagonists inhibited the binding of [3H]bradykinin to Chaps-solubilized extracts with relative potencies similar to those observed with the low-affinity membrane-bound binding sites. Following partial purification of the soluble preparation, using anion exchange (DEAE-Sephacel) and gel filtration (Aca 34) column chromatography steps, two peaks eluted off the column were able to bind [3H]bradykinin and have molecular masses of 168 and 98.5 kDa. The former seems to represent binding of bradykinin to angiotensin converting enzyme (ACE, EC 3.4.15.1) and the latter binding to bradykinin receptor. Using purified commercial ACE, we show that the binding of [3H]bradykinin to ACE can easily be distinguished from that of the bradykinin receptor, since both B1 and B2 ligands were able to inhibit bradykinin binding with affinities clearly different from that expected for a bradykinin receptor.
Trifilieff
A
A
Laboratorie de Neuroimmunopharmacologie INSERM CJF 91-05, Université Louis Pasteur-Strasbourg I, Illkirch, France.
Lach
E
E
Mousli
M
M
Haddad
E B
EB
Landry
Y
Y
Gies
J P
JP
D000818:Animals; D001665:Binding Sites; D002793:Cholic Acids; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents; D006168:Guinea Pigs; D007700:Kinetics; D008024:Ligands; D008168:Lung / Q000201:enzymology / Q000378:metabolism*; D008566:Membranes / Q000201:enzymology / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D018002:Receptors, Bradykinin / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D008024:Ligands; D018002:Receptors, Bradykinin; D007703:Peptidyl-Dipeptidase A; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate