Spatial organization of CaATPase molecules in sarcoplasmic reticulum vesicles.
Fluorescence intensity, polarization, and (Ca2+-Mg2+)-ATPase (CaATPase) activity were measured for sarcoplasmic reticulum (SR) CaATPase with varying amounts of fluorescein isothiocyanate (FITC) attached at a specific site at or near the ATP binding site. The stoichiometry of attached FITC was proportional to the inhibition of ATPase activity, consistent with the independent labeling of one FITC site per CaATPase molecule. Polarization measurements on vesicular CaATPase indicated the occurrence of energy-transfer depolarization that increased as the fraction of binding sites labeled by FITC increased. Addition of the nonionic detergent dodecyl nonaoxyethylene alcohol (C12E9) eliminated the energy-transfer depolarization for all degrees of labeling with little direct effect on the attached FITC molecule. Fluorescence polarization measurements on sizing-column-purified FITC-labeled CaATPase in the presence of 30 mM C12E9 indicated that the sample consisted of homogeneous monomeric CaATPase. The attached FITC molecule was not sensitive to the bulk viscosity for either the vesicular or the detergent-solubilized CaATPase. The midpoints of the transition from vesicular to monomeric CaATPase as a function of increasing detergent concentration were determined from fluorescence polarization and light-scattering measurements. The dependence of these midpoints on the CaATPase concentration indicated a stoichiometry of 262 +/- 35 molecules of C12E9 per CaATPase in the detergent-protein complex. Both measurements gave the same result. The decrease of fluorescence polarization with increasing saturation of the FITC binding sites for vesicular and detergent-solubilized CaATPase was analyzed in terms of energy-transfer depolarization to determine the spatial arrangements of CaATPase molecules.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013861:Thiocyanates
D005452:Fluoresceins; D013861:Thiocyanates; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate