title: Halothane and cyclopiazonic acid modulate Ca-ATPase oligomeric state and function in sarcoplasmic reticulum.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
authors.forename: B S
authors.initials: BS
authors.lastname: Karon
authors.forename: J E
authors.initials: JE
authors.lastname: Mahaney
authors.forename: D D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: 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
chemical_list: D007211:Indoles; D008563:Membrane Lipids; D010710:Phosphates; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium; D006221:Halothane; C000543:cyclopiazonic acid