These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Detergent solubilization, functional reconstitution, and partial purification of epithelial amiloride-binding protein.
    Author: Sariban-Sohraby S, Benos DJ.
    Journal: Biochemistry; 1986 Aug 12; 25(16):4639-46. PubMed ID: 3768303.
    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 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)
    [Abstract] [Full Text] [Related] [New Search]