Halothane and cyclopiazonic acid modulate Ca-ATPase oligomeric state and function in sarcoplasmic reticulum.
We have studied the effects of cyclopiazonic acid (CPA) and halothane on the enzymatic activity, oligomeric state, and conformational equilibrium of the Ca-ATPase in skeletal muscle sarcoplasmic reticulum (SR). CPA is a potent inhibitor of Ca-ATPase activity, and this inhibition is competitive with respect to ATP concentration. Time-resolved phosphorescence anisotropy was used to detect the fraction of Ca-ATPase monomers, dimers, and larger aggregates in the absence and presence of CPA. CPA increased the fraction of dimers and larger aggregates of the Ca-ATPase. Addition of halothane to SR, or detergent solubilization of the Ca-ATPase, increased the apparent KI of CPA inhibition, and increased the fraction of Ca-ATPase present as monomers. CPA stabilized the E2 conformational state of the Ca-ATPase relative to the E1 and E2-P states, as measured by fluorescein 5-isothiocyanate fluorescence and enzyme phosphorylation from inorganic phosphate. E2-P formation in the presence of CPA was partially restored by halothane and solubilization. We conclude that CPA inhibits the Ca-ATPase in part by overstabilizing dimers or small oligomers of the Ca-ATPase, which is correlated with stabilization of the E2 conformation of the enzyme.
Karon
B S
BS
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Mahaney
J E
JE
Thomas
D D
DD
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005454:Fluorescence Polarization; D006221:Halothane / Q000494:pharmacology*; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology*; D008560:Membrane Fluidity; D008563:Membrane Lipids / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology*; D013329:Structure-Activity Relationship
D007211:Indoles; D008563:Membrane Lipids; D010710:Phosphates; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium; D006221:Halothane; C000543:cyclopiazonic acid