Detergent solubilization, functional reconstitution, and partial purification of epithelial amiloride-binding protein.
The amiloride-binding protein from cultured toad kidney cells (A6) was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), functionally reconstituted into liposomes, and partially purified. The specific binding of [3H]methylbromoamiloride ([3H]CH3BrA) was measured in intact A6 epithelia, A6 cell homogenate (H), apical plasma membrane vesicle (V1), and CHAPS-solubilized V1 and on material obtained after affinity chromatography of CHAPS-solubilized plasma membrane vesicles on agarose-immobilized wheat germ agglutinin (WGA). Specific [3H]CH3BrA binding to H, V1, and WGA material reached equilibrium after 10 min. Scatchard analysis of [3H]CH3BrA binding to V1 and WGA material revealed a homogeneous class of binding sites with KD's of 130 and 128 nM, respectively. These KD values were similar to the apparent inhibitory dissociation constant determined from amiloride inhibition of 22Na+ influx in both intact A6 epithelia and V1. The total number of specific binding sites was 4 pmol/mg of V1 protein, which represented a 10-fold enrichment compared to H, and 66.6 pmol/mg of WGA material (a 148-fold enrichment). From association/displacement kinetic studies of specific [3H]CH3BrA binding to V1, the rate constants of association (ka) and dissociation (kd) were calculated to be 3.6 X 10(5) M-1 s-1 and 49.5 X 10(-3) s-1, respectively. These values yield an equilibrium dissociation constant of 138 nM. In solubilized V1 protein, binding activity was enriched approximately 20-fold over H and was markedly dependent upon the relative concentrations of detergent and phospholipid. CHAPS solubilization of V1 resulted in an average 44% recovery of protein with 90% retention of the total number of specific [3H]CH3BrA binding sites. After WGA chromatography 2.7% of the applied protein and 46% of the specific binding sites were recovered.(ABSTRACT TRUNCATED AT 250 WORDS)
D000584:Amiloride / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids; D003902:Detergents; D004848:Epithelium / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D012964:Sodium / Q000378:metabolism
D002352:Carrier Proteins; D002793:Cholic Acids; D003902:Detergents; D000584:Amiloride; D012964:Sodium; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate