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pubmed:abstractText |
Fluoroaluminate in combination with nucleotide inhibited ATPase activity of P-glycoprotein (Pgp) in plasma membranes and in pure reconstituted form. Low nucleotide concentrations were effective, e.g., half-maximal inhibition was obtained with 10 microM MgATP. With MgATP or MgADP, reactivation occurred with t1/2 = 7 min at 37 degrees C. With 8-azido-ATP, UV irradiation of inhibited Pgp gave specific photolabeling of both nucleotide sites. Fluoroaluminate therefore provides a valuable tool for functional and structural characterization of P-glycoprotein and probably of other ABC transporters. 2-Azido-ATP, in combination with vanadate, fluoroaluminate, or beryllium fluoride, inhibited Pgp ATPase activity. Low concentrations of 2-azido-ATP were effective. However, after UV irradiation of the inhibited Pgp species, in no case was there evidence of covalent labeling of nucleotide sites. Therefore in the Pgp catalytic sites, under conditions of nucleotide trapping, there is no suitable amino acid side chain adjacent to the photoactivated 2-position of bound 2-azido-nucleotide, and 8-azido-ATP is the preferred photolabeling analog.
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