Characteristics of the Interaction of Melittin with Sarcoplasmic Reticulum Membranes

E. A. Shorina¹, N. V. Mast¹, K. B. Storey², O. D. Lopina¹, and A. M. Rubtsov^1*

¹Department of Biochemistry, School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-3955; E-mail: amr@atpase.bio.msu.su

²Department of Biochemistry and Department of Chemistry, Carleton University, Ottawa K1S 5B6, Canada; fax: (613) 520-4497; E-mail: kbstorey@ccs.carleton.ca

^* To whom correspondence should be addressed.

Received August 21, 1998; Revision received September 23, 1998

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Addition of an amphipathic bee venom peptide, melittin, to sarcoplasmic reticulum (SR) vesicles isolated from rabbit skeletal muscles resulted in a fast (<1 min) blue shift in the fluorescence maximum of the melittin--SR membrane complex. Over the following 45 min the position of the fluorescence maximum did not change, but the fluorescence intensity of the melittin--SR membrane complex decreased by ~35% with rate constant 0.14 min^-1. Melittin rapidly quenched the isotropic signal in the EPR spectrum of spin-labeled stearic acid added to SR membranes. Further changes in the spectral parameters of the spin probe bound to SR membranes in the presence of melittin indicated an increase of the viscosity of the probe microenvironment (empiric parameter T/eta was decreased by ~35% with rate constant 0.11 min^-1). The surface potential of SR membranes measured using a pH-sensitive dye, neutral red, decreased after melittin addition from -60 to -30 mV. It was demonstrated with the use of a cross-linking agent, cupric o-phenanthroline, that melittin induced slow aggregation of Ca-ATPase protein in SR membranes; the content of enzyme in the monomeric form decreased with rate constant 0.14 min^-1. It is concluded that melittin binds rapidly to SR membranes, inducing slow changes in Ca-ATPase conformation and oligomeric state as well as structural transitions in the lipid bilayer of SR membranes.

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KEY WORDS: sarcoplasmic reticulum, Ca-ATPase, melittin, fluorescence, EPR spectroscopy, cross-linking agents

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