Binding and degradation of angiotensin II by mesenteric artery subcellular membranes.
Rat mesenteric artery microsomes were previously reported to degrade 125I-angiotensin II (AII). It is now shown here that washing the membranes with EDTA and including EGTA in the assay media reduces the 125I-AII degradation to very low levels without reducing the specific binding of 125I-AII. Using EDTA wash and including 5 mM MgCl2 and 0.2 mM EGTA the following characteristics of the binding were observed: microscopic association rate constant (k1) = 1.3 to 2.2 X 10(5) M-1 s-1, microscopic dissociation rate constant (k-1) = 3.8 to 6.4 X 10(-4) s-1, equilibrium dissociation constant (Kd) = 1.8 nM and number of binding sites (Bmax) = 193 fmol/mg protein. The subcellular distribution of the specific binding of 125I-AII at 0.16 nM and 1.63 nM was studied along with the distribution of the marker enzymes. The specific binding paralleled the plasma membrane marker (5'-nucleotidase), but not the putative endoplasmic reticulum marker (NADPH-cyt. c-reductase) or the inner mitochondrial marker (cyt. c-oxidase). Thus the binding to the plasma membrane-enriched fraction F2 occurred with a similar affinity (Kd = 2.2 nM) but with higher number of binding sites (420 fmol/mg protein). This study establishes the 125I-AII binding method suitable for determining the changes in the angiotensin receptor characteristics in the pathophysiology of the vascular smooth muscle.
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D004533:Egtazic Acid / Q000494:pharmacology; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008638:Mesenteric Arteries / Q000378:metabolism / Q000648:ultrastructure; D008861:Microsomes / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D011945:Receptors, Angiotensin / Q000378:metabolism
D007457:Iodine Radioisotopes; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D004533:Egtazic Acid; D008274:Magnesium