Effects of melittin on molecular dynamics and calcium-ATPase activity in sarcoplasmic reticulum membranes: time-resolved optical anisotropy

Abstract

We have studied the effect of melittin, a basic membrane-binding peptide, on Ca-ATPase activity and on protein and lipid dynamics in skeletal sarcoplasmic reticulum (SR), using time-resolved phosphorescence and fluorescence spectroscopy. Melittin completely inhibits Ca-ATPase activity, with half-maximal inhibition at 9 +/- 1 mol of melittin bound to the membrane per mole of ATPase (0.1 mol of melittin per mole of lipid). The time-resolved phosphorescence anisotropy (TPA) decay of the Ca-ATPase labeled with erythrosin isothiocyanate (ERITC) shows that melittin restricts microsecond protein rotational motion. At 25 degrees C in the absence of melittin, the TPA is characterized by three decay components, corresponding to a rapid segmental motion (correlation time phi 1 = 2-3 microseconds), the uniaxial rotation of monomers or dimers (phi 2 = 16-22 microseconds), and the uniaxial rotation of larger oligomers (phi 3 = 90-140 microseconds). The effect of melittin is primarily to decrease the fraction of the more mobile monomer/dimer species (A2) while increasing the fractions of the larger oligomer (A3) and very large aggregates (A infinity). Time-resolved fluorescence anisotropy of the lipid-soluble probe diphenylhexatriene (DPH) shows only a slight increase in the lipid hydrocarbon chain effective order parameter, corresponding to an increase in lipid viscosity that is too small to account for the large decrease in protein mobility or inhibition of Ca-ATPase activity. Thus the inhibitory effect of melittin correlates with its capacity to aggregate the Ca-ATPase and is consistent with previously reported inhibition of this enzyme under conditions that increase protein-protein interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Melittin: a Membrane-active Peptide with Diverse Functions

“…For instance, it has been shown that lytic concentrations of melittin dramatically reduce the rotational mobility of band 3 protein in human erythrocyte membranes (Dufton et al 1984;Clague and Cherry 1988;Hui et al 1990) and of bacteriorhodopsin in lipid vesicles (Hu et al 1985). Further, melittin causes aggregation of membrane proteins including band 3 protein (Clague and Cherry 1988), bacteriorhodopsin (Hu et al 1985) and Ca 2+ -ATPase (Voss et al 1991;Mahaney and Thomas 1991;Mahaney et al 1992). In addition, melittin is a potent inhibitor of Ca 2+ -ATPase (Voss et al 1991;Mahaney and Thomas, 1991), H + K + -ATPase (Cuppoletti et al 1989;Cuppoletti, 1990) and Na + K + -ATPase (Cuppoletti and Abbott, 1990).…”

“…Further, melittin causes aggregation of membrane proteins including band 3 protein (Clague and Cherry 1988), bacteriorhodopsin (Hu et al 1985) and Ca 2+ -ATPase (Voss et al 1991;Mahaney and Thomas 1991;Mahaney et al 1992). In addition, melittin is a potent inhibitor of Ca 2+ -ATPase (Voss et al 1991;Mahaney and Thomas, 1991), H + K + -ATPase (Cuppoletti et al 1989;Cuppoletti, 1990) and Na + K + -ATPase (Cuppoletti and Abbott, 1990). The experiments with bacteriorhodopsin suggest that immobilization due to aggregation of membrane proteins is a result of direct melittin-protein interactions rather than an indirect consequence of melittin-lipid interactions (Hu et al 1985).…”

Dissociation of Phospholamban Regulation of Cardiac Sarcoplasmic Reticulum Ca2+ATPase by Quercetin

“…Ca 2ϩ ATPase transports two Ca 2ϩ ions across the SR membrane against a large concentration gradient by hydrolytic coupling with one ATP molecule. Structural studies (Toyoshima et al, 1993;Stokes et al, 1994) suggest that Ca 2ϩ ATPase molecules can exist in monomeric and oligomeric forms with enzymatic activity being regulated by its oligomeric state (Squier et al, 1988;Voss et al, 1991Voss et al, , 1994Kutchai et al, 1994). Large aggregates of enzyme have low or no activity, and dissociated enzyme (possibly dimers) has high activity.…”

Effects of Photo-oxidizing Analogs of Fluorescein on the Sarcoplasmic Reticulum Ca2+-ATPase

“…However, Murphy's data and our own results show that the Ca 2ϩ -ATPase affinity for free fluorescein is not particularly high, being strongly increased (at least two orders of magnitude) by the addition of halogenated substitutes to the molecule. Many authors have used eosin isothiocyanate (Papp et al, 1987;Munkonge et al, 1988) and erythrosin isothiocyanate (Papp et al, 1987;Birmachu and Thomas, 1990;Voss et al, 1991) rather than FITC, due to their higher affinity and particular fluorescence characteristics, to study rotational movements of the SR Ca 2ϩ -ATPase. These authors concluded that both compounds bind to the same site and react with the same lysyl residue as FITC (Papp et al, 1987;Birmachu and Thomas, 1990).…”