INHIBITION OF P-GLYCOPROTEIN ATPASE ACTIVITY BY PROCEDURES INVOLVING TRAPPING OF NUCLEOTIDE IN CATALYTIC SITES

Fluoroaluminate in combination with nucleotide inhibited ATPase activi ty of P-glycoprotein (Pgp) in plasma membranes and in pure reconstitut ed form. Low nucleotide concentrations were effective, e.g., half-maxi mal inhibition was obtained with 10 mu M MgATP. With MgATP or MgADP, r eactivation occurred with t(1/2) = 7 min at 37 degrees C. With 8-azido -ATP, UV irradiation of inhibited Pgp gave specific photolabeling of b oth nucleotide sites. Fluoroaluminate therefore provides a valuable to ol for functional and structural characterization of P-glycoprotein an d probably of other ABC transporters. 2-Azido-ATP, in combination with vanadate, fluoroaluminate, or beryllium fluoride, inhibited Pgp ATPas e activity. Low concentrations of 2-azido-ATP were effective. However, after UV irradiation of the inhibited Pgp species, in no case was the re evidence of covalent labeling of nucleotide sites. Therefore in the Pgp catalytic sites, under conditions of nucleotide trapping, there i s no suitable amino acid side chain adjacent to the photoactivated a-p osition of bound 2-azido-nucleotide, and 8-azido-ATP is the preferred photolabeling analog. (C) 1997 Academic Press.