Inhibition of the sarcoplasmic reticulum Ca2+-ATPase by Reactive Red 120.
The triazine dye, Reactive Red 120, was found to bind tightly (Kd = 30) nM) and with low stoichiometry to sarcoplasmic reticulum membranes. Our finding that this high-affinity binding caused noncompetitive inhibition of the Ca2+-ATPase indicates that the dye-binding site is distinct from both the active site and putative regulatory site. Detergent solubilization (monomerization) of the Ca2+-ATPase caused a 25-fold decrease in affinity for Reactive Red 120, while causing no decrease in affinity toward another dye, Reactive Blue 2. For the Reactive-Red-120-inhibited enzyme, the level of steady-state enzyme phosphorylation by ATP was not significantly different from that exhibited by the control Ca2+-ATPase. The rate of dephosphorylation in the presence and absence of ADP, however, was markedly decreased by the presence of the inhibitor. Distance measurements by fluorescence energy transfer from the active (FITC-reactive) site to the Reactive Red 120 site gave a value of 59 A. Similar experiments yielded an average distance of 35 A between the latter site and the tryptophan residues, most of which are postulated by the 'sequence model' (MacLennan et al. (1985) Nature 316, 696-700) to be located in a transmembrane domain.
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D004396:Coloring Agents / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009132:Muscles / Q000201:enzymology*; D010766:Phosphorylation; D011770:Pyruvate Kinase / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014227:Triazines
D004396:Coloring Agents; D003902:Detergents; D014227:Triazines; C042516:reactive red 120 dye; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase; D000252:Calcium-Transporting ATPases