diphenylhexatriene and erythrosine-isothiocyanate

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)

Topics: Animals; Calcium-Transporting ATPases; Diphenylhexatriene; Erythrosine; Fluorescence Polarization; Isothiocyanates; Kinetics; Macromolecular Substances; Melitten; Muscles; Polylysine; Rabbits; Sarcoplasmic Reticulum; Spectrometry, Fluorescence

The rotational dynamics of the purified dicyclohexylcarbodiimide-sensitive H(+)-ATPase (DSA) reconstituted into phospholipid vesicles and of the DSA coreconstituted with the proton pump bacterio-rhodopsin were examined by using the technique of time-resolved phosphorescence emission anisotrophy. The phosphorescent probe erythrosin isothiocyanate was used to covalently label the gamma-polypeptide of DSA before reconstitution. Rotational correlation times were measured under a variety of conditions. The rotational correlation time was independent of the viscosity of the external medium but increased significantly as the microviscosity of the membrane increased. This indicates the rotational correlation times are a measure of the enzyme motion within the membrane. The activation energy associated with the rotational correlation time is 8-10 kcal/mol. At 4 degrees C, the correlation time, typically approximately 100-180 microseconds, was unaffected by the addition of substrates and the presence of a membrane pH gradient. Therefore, molecular rotation of the DSA does not appear to play an important role in enzyme catalysis or ion pumping.

Topics: Chloroplasts; Diphenylhexatriene; Erythrosine; Isothiocyanates; Molecular Probes; Phospholipids; Plants; Proton-Translocating ATPases; Spectrometry, Fluorescence