Mechanism of Ca-ATPase inhibition by melittin in skeletal sarcoplasmic reticulum.
We have previously shown that the basic, amphipathic peptide melittin inhibits the Ca-ATPase of the sarcoplasmic reticulum membrane by inducing large-scale aggregation of the enzyme via electrostatic cross-linking. To better understand the physical mechanism by which melittin-induced Ca-ATPase aggregation inhibits the enzyme, we have performed time-resolved phosphorescence anisotropy (TPA) and steady-state fluorescence experiments in combination with enzyme kinetic assays, utilizing (1) native and charge-modified melittin in order to characterize the peptide charge dependence of the melittin-SR interaction, and (2) various calcium levels in order to define the effect of melittin on the enzyme's E1 and E2 conformational equilibrium. TPA results showed that decreasing melittin's positive charge dramatically decreases the ability of the peptide to aggregate the enzyme, which correlates with a reduced potency of the modified peptide to inhibit enzymatic activity. Steady-state fluorescence of fluorescein isothiocyanate-labeled Ca-ATPase showed that melittin reduces Ca-ATPase affinity for calcium by shifting the enzyme's E1-E2 conformational equilibrium toward E2, but increasing calcium progressively reverses this shift. Kinetic experiments showed that melittin does not prevent ATP-dependent enzyme phosphorylation, but it completely inhibits Pi-dependent EP formation and substantially slows Pi release during steady-state cycling. We conclude that melittin-induced aggregation of the Ca-ATPase depends on the electrostatic interaction of the peptide with cytoplasmic Ca(2+)-dependent sites on the enzyme, and that enforced Ca-ATPase protein-protein interactions inhibit the conformational transitions that facilitate phosphoenzyme hydrolysis.
Voss
J C
JC
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Mahaney
J E
JE
Thomas
D D
DD
D000107:Acetylation; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008555:Melitten / Q000378:metabolism / Q000494:pharmacology*; D009038:Motion; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013329:Structure-Activity Relationship; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D008555:Melitten; D000099211:Phospholamban