PMID: 3768303Aug 12, 1986Paper

Detergent solubilization, functional reconstitution, and partial purification of epithelial amiloride-binding protein

Biochemistry
S Sariban-Sohraby, D J Benos

Abstract

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 con...Continue Reading

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Feb 1, 1986·The American Journal of Physiology·S Sariban-Sohraby, D J Benos
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Mar 1, 1982·The American Journal of Physiology·D J Benos

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Citations

Oct 1, 1988·The Journal of Membrane Biology·T R Kleyman, E J Cragoe
Nov 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·D J BenosS Sariban-Sohraby
Jul 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·P BarbryM Lazdunski
Aug 15, 1991·Proceedings of the National Academy of Sciences of the United States of America·P R SmithD J Benos
Aug 1, 1992·Kidney International·C P Bastl, J P Hayslett
Dec 1, 1987·Kidney International·C FrelinM Lazdunski
Oct 1, 1990·Kidney International·M Martinez-Maldonado, H R Cordova
May 1, 1988·Journal of Cellular Physiology·M Konieczkowski, M J Dunn
Jan 14, 1999·The American Journal of Physiology·S Sariban-SohrabyF Mies
Aug 7, 1999·The Journal of Biological Chemistry·J B ZuckermanP R Smith
Oct 22, 2002·American Journal of Physiology. Renal Physiology·Vadim G ShlyonskySarah Sariban-Sohraby
May 11, 1992·The American Journal of Physiology·S MatalonD J Benos
Apr 1, 1988·The American Journal of Physiology·R J BridgesW Rummel
Dec 1, 1988·The American Journal of Physiology·E J SorscherD J Benos
Sep 1, 1995·The American Journal of Physiology·A L BradfordD J Benos
Nov 1, 1992·The American Journal of Physiology·S Sariban-SohrabyR S Fisher
Jan 11, 1989·The American Journal of Physiology·R J BridgesD J Benos
Jul 1, 1995·The American Journal of Physiology·A PuotiB C Rossier

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