Equilibrium and kinetic fluorescence methods have been used to characterize the interactions between K+ and the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum. K+ shifts the E2-E1 equilibrium of the ATPase towards E1 and increases the rate of Ca2+ binding to the ATPase, as detected by changes in tryptophan fluorescence intensity, suggesting that K+ increases the rate of the E2-E1 transition. The data are consistent with binding of K+ at the inner Ca(2+)-binding site on the ATPase in competition with H+ and Mg2+, with a higher affinity in the E1 than in the E2 conformation. K+ has no effect on the affinity for Mg2+, as detected by changes in tryptophan fluorescence intensity; since it has been proposed that the changes in tryptophan fluorescence follow from binding to Mg2+ at the outer Ca(2+)-binding site, this suggests that K+ is unable to bind at the outer Ca(2+)-binding site. K+ increases the rate of dissociation of Ca2+ from the Ca(2+)-bound ATPase and reduces the effect of Mg2+ on the fluorescence intensity of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It is suggested that these effects of K+ are the result of binding at a 'gating' site on the ATPase, in competition with binding of H+. Binding of K+ at the inner Ca(2+)-binding site and at the gating site account for the observed effects of K+ on the affinity of the ATPase for Ca2+.

The steady-state ATPase activity of sarcoplasmic-reticulum (Ca2+-Mg2+)-ATPase is inhibited by thapsigargin at a molar ratio of 1:1, with a dissociation constant for thapsigargin estimated to be in the sub-nanomolar range. In the presence of thapsigargin, only a single Ca2+ ion binds to the ATPase. Similarly, addition of thapsigargin to the ATPase incubated in the presence of Ca2+ results in the release of one of the two originally bound Ca2+ ions. As monitored by the fluorescence of nitrobenzo-2-oxa-1,3-diazole-labelled ATPase, thapsigargin appears to shift the transition between El and E2 conformations towards E2. Addition of thapsigargin prevents phosphorylation of the ATPase by Pi and results in a very low steady-state level of phosphorylation of the ATPase by ATP, as observed previously for nonylphenol.

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum has been reconstituted with peptides corresponding to the hydrophobic domain of phospholamban (PLB) with or without the three Cys residues replaced by Ala, and with PLB with the three Cys residues replaced by Ala [PLBcys-(1-52)]. Reconstitution with the hydrophobic domain of PLB[PLB(25-52)] was found to decrease the apparent affinity of the ATPase for Ca2+ with no effect on the maximal rate of ATP hydrolysis observed at saturating concentrations of Ca2+. Reconstitution with PLBCys-(1-52) decreased both the apparent affinity for Ca2+ and the maximal activity; the effect on maximal activity followed from a decrease in the rate of the Ca2+ transport step (E1PCa2-->E2P) as observed with the hydrophilic domain PLB(1-25). The concentration dependences of the effects of the hydrophobic domain and of the whole PLB molecule were very similar, suggesting that the hydrophilic domain made little contribution to the affinity of the ATPase for PLB. The effect of PLB on the ATPase was dependent on the molar ratio of phospholipid to ATPase, suggesting partition of the PLB between its binding site on the ATPase and the bulk lipid phase in the membrane. Neither PLB nor its hydrophobic domain affected the rates of phosphorylation or dephosphorylation of the ATPase. Despite their effects on the apparent affinity of the ATPase for Ca2+, neither PLB nor its hydrophobic domain had any effect on the true affinity of the ATPase for Ca2+, as measured from changes in the tryptophan fluorescence of the ATPase. The effects of PLB on the activity of the ATPase are the sum of the effects of its hydrophilic and hydrophobic domains.

We have studied the effects of aromatic residues at the ends of peptides of the type Ac-KKGL(n)()WL(m)()KKA-amide on their interactions with lipid bilayers as a function of lipid fatty acyl chain length, physical phase, and charge. Peptide Ac-KKGFL(6)WL(8)FKKA-amide (F(2)L(14)) incorporated into bilayers of phosphatidylcholines containing monounsaturated fatty acyl chains of lengths C14-C24 at a peptide:lipid molar ratio of 1:100 in contrast to Ac-KKGL(7)WL(9)KKA-amide (L(16)) which did not incorporate at all into dierucoylphosphatidylcholine [di(C24:1)PC]; Ac-KKGYL(6)WL(8)YKKA-amide (Y(2)L(14)) incorporated partly into di(C24:1)PC. Lipid-binding constants relative to that for dioleoylphosphatidylcholine (C18:1)PC were obtained using a fluorescence quenching method. For Y(2)L(14) and F(2)L(14), relative lipid-binding constants increased with increasing fatty acyl chain length from C14 to C24; strongest binding did not occur at the point where the hydrophobic length of the peptide equalled the hydrophobic thickness of the bilayer. For Ac-KKGYL(9)WL(11)YKKA-amide (Y(2)L(20)), increasing chain length from C18 to C24 had little effect on relative binding constants. Anionic phospholipids bound more strongly than zwitterionic phospholipids to Y(2)L(14) and Y(2)L(20) but effects of charge were relatively small. In two phase (gel and liquid crystalline) mixtures, all the peptides partitioned more strongly into liquid crystalline than gel phase; effects were independent of the structure of the peptide or of the lipid (dipalmitoylphosphatidylcholine or bovine brain sphingomyelin). Addition of cholesterol had little effect on incorporation of the peptides into lipid bilayers. It is concluded that the presence of aromatic residues at the ends of transmembrane alpha-helices effectively buffers them against changes in bilayer thickness caused either by an increase in the chain length of the phospholipid or by the presence of cholesterol.

We have developed a procedure for the reconstitution of Escherichia coli diacylglycerol kinase (DGK) into phospholipid bilayers containing diacylglycerol substrate. When DGK is reconstituted into a series of phosphatidylcholines containing monounsaturated fatty acyl chains, activity against dihexanoylglycerol (DHG) as a substrate was found to be markedly dependent on the fatty acyl chain length with the highest activity in dioleoylphosphatidylcholine [di(C18:1)PC] and a lower activity in bilayers with shorter or longer fatty acyl chains. Low activities in the short chain phospholipid dimyristoleoylphosphatidylcholine [di(C14:1)PC] followed from an increase in the K(m) value for DHG and ATP, with no effect on v(max). In contrast, in the long chain lipid dierucoylphosphatidylcholine [di(C24:1)PC], the low activity followed from a decrease in v(max) with no effect on K(m). In mixtures of two phosphatidylcholines with different chain lengths, the activity corresponded to that expected for the average chain length of the mixture. Cholesterol increased the activity in di(C14:1)PC but slightly decreased it in di(C18:1)PC or di(C24:1)PC, effects that could follow from changes in bilayer thickness caused by cholesterol.