MECHANISM OF INHIBITION OF THE PLASMA-MEMBRANE CA2-ATPASE BY BARBITURATES()

We have demonstrated that sodium pentobarbital inhibited the activatio n of the human red blood cell plasma membrane Ca2+-ATPase produced by dimerization of enzyme monomers or by calmodulin binding to enzyme mon omers. The effects of the barbiturate were dose-dependent. Both V-max and Ca2+ affinity were reduced. The Ca2+-ATPase activity of the dimeri c enzyme was distinctly less sensitive with respect to the effective i nhibitory concentrations of pentobarbital and to the rate of onset of inhibition than was the calmodulin-dependent activation of enzyme mono mers. Temperature dependence of the inhibition was in agreement with d irect, nonpolar interactions of pentobarbital with a water-exposed non polar patch on the surface of this transmembrane protein. The barbitur ate prevented the increase of intrinsic tryptophan fluorescence associ ated with substrate Ca2+ binding to the enzyme dimer, On the basis of the barbiturate effects we propose a model for the action of detergent -like compounds on the enzyme, They inhibit Ca2+-ATPase activity by bi nding to a nonpolar patch on the water-exposed dimerization surface of the enzyme monomer, part of which is also the binding site for calmod ulin. The model assumes that their binding to the nonpolar patch on th e monomer interferes with dimerization and weakens but does not prohib it calmodulin binding, whose activation of the enzyme is then submaxim al. The model should be applicable to other proteins as the two activa tion pathways studied have been demonstrated for various enzymes.