title: Binding of the fluorescent substrate analogue 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine 5'-triphosphate to the gastric H+,K(+)-ATPase: evidence for cofactor-induced conformational changes in the enzyme.
abstract: TNP-ATP binds to the gastric H,K-ATPase with a 4.6-fold increase in fluorescence intensity and 10-nm blue shift that indicate a relatively hydrophobic protein environment. The fluorescence enhancement saturates and is compatible with binding to a single class of specific nucleotide sites with Kd less than 25 nM and N = 3.4 +/- 0.9 nmol mg-1. Cofactors of the H,K-ATPase affect the fluorescence enhancement. K+ causes a rapid fluorescence quench by binding to a single class of sites with Kd = 3 mM. Mg2+ rapidly and completely reverses the K+ quench and then causes a slow fluorescence quench. The maximum enhancement is approximately halved by either Mg2+ or K+ in titrations with both protein and fluorophore. Therefore, TNP-ATP reports changes in protein environment compatible with cofactor-induced changes in the conformation of the enzyme.
authors.affiliation: Department of Medicine, University of California, Los Angeles School of Medicine, California.
authors.forename: L D
authors.initials: LD
authors.lastname: Faller
mesh_terms: D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000818:Animals; D001051:Apoenzymes; D001059:Apoproteins; D002413:Cations, Divalent / Q000494:pharmacology*; D005456:Fluorescent Dyes; D017506:H(+)-K(+)-Exchanging ATPase; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation; D013270:Stomach / Q000187:drug effects / Q000201:enzymology
chemical_list: D001051:Apoenzymes; D001059:Apoproteins; D002413:Cations, Divalent; D005456:Fluorescent Dyes; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D008274:Magnesium; D011188:Potassium