Molecular topography of the Ca2+-ATPase of sarcoplasmic reticulum.
The Ca2+ binding site region of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum was labeled with several fluorescent analogs of dicyclohexylcarbodiimide. As has been shown by Chadwick and Thomas, in the absence of Ca2+ in the medium, labeling with the naphthyl carbodiimide results in the inhibition of enzyme activity. Further, Ca2+ occupancy of the high affinity sites of the enzyme protects against incorporation into the site(s). The fluorescent carbodiimide has been used to determine the depth of the site of label incorporation relative to the aqueous-bilayer interfaces by quenching studies using spin-labeled fatty acid derivatives. The series of quenchers used have their spin-label moiety located at different positions along the fatty acid chain. It was found that after suitable correction for differences in partitioning of the various derivatives, the order of quenching efficiency was 16- greater than 12- greater than 10- greater than 7- greater than 5-NS, indicating that the naphthyl moiety is near the center of the bilayer. In contrast, quenching with the aqueous-restricted I- indicated that the label is accessible from the external milieu, likewise for a presumed aqueous quencher, acrylamide. The aqueous quenchers accessibilities were altered upon Ca2+ binding to the ATPase. Quenching of the intrinsic fluorescence with the x-NS derivatives indicates that the ATPase tryptophan residues are primarily localized at the aqueous-membrane interfaces, with the order of quenching being 5- greater than 7- greater than 10- greater than 12- greater than 16-NS. The trp residue(s) which changes its fluorescence upon Ca2+ binding is shown to be near the membrane surface.
Scott
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Boston Biomedical Research Institute, MA 02114.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D000252:Calcium-Transporting ATPases; D002118:Calcium