MECHANISM OF INHIBITION OF THE CA2-ATPASE BY MELITTIN()

The Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by melittin at pH 7.4. Melittin has no effect on the rate of phosphor ylation of the ATPase or on the rate of the Ca2+ transport step, but m elittin inhibits dephosphorylation of the phosphorylated ATPase at pH 7.3. At pH 6.0, melittin has no effect on ATPase activity or on the ra te of dephosphorylation. At pH 7.4, inhibition of ATPase activity fitt ed to a K-d of 0.4 mu M for melittin. Analogues of melittin in which t he two Arg residues were replaced by Gln [melittin(RR to QQ)] or the t wo Lys residues were replaced by Gln [melittin-(KK to QQ)] also inhibi ted ATPase activity, but with an increased K-d value of 3.4 mu M. Anal ogues of melittin containing an extra Lys residue at the C-terminus [m elittin(+K)] or in which the Trp residue had been replaced with a Leu residue [melittin(W to L)] had the same effect on activity as melittin . Melittin and all the analogues increased the permeability of the SR membrane to Ca2+ with equal potency at pH 6.4, as shown by a reduction in level of Ca2+ accumulation. Melittin and all the analogues also sh ifted the E2-E1 equilibrium of the ATPase toward E1 with equal potency at pH 7.2, consistent with stronger binding to the E1 conformation. I t is suggested that effects on Ca2+ permeability and on the E2-E1 equi librium could follow from binding of the N-terminus of melittin at the membrane-water interface, and that effects on ATPase activity could f ollow from binding of the positively charged C-terminus between the ph osphorylation and nucleotide binding domains. Inhibition of ATPase act ivity by melittin is observed in reconstituted vesicles containing sin gle ATPase molecules. Binding of monoclonal antibodies to the ATPase d oes not prevent inhibition of ATPase activity by melittin. We conclude that inhibition does not require aggregation of the ATPase molecules.