(Iodoacetamido)fluorescein labels a pair of proximal cysteines on the Ca2+-ATPase of sarcoplasmic reticulum.
Previous energy transfer studies [Squier, T. C., Bigelow, D. J., de Ancos, J. G., & Inesi, G. (1987) J. Biol. Chem. 262, 4748-4754] have utilized fluorescent iodoacetamide derivatives covalently bound to the Ca2+-ATPase of sarcoplasmic reticulum (SR), using labeling conditions that completely modify the most reactive of the protein's surface sulfhydryls to a final level of 9 nmol/mg of SR protein. Unambiguous interpretation of these results requires localization of these labeling sites with respect to the primary structure of the Ca2+-ATPase. In the present study, we have used the probe 6-(iodoacetamido)fluorescein (IAF) as a marker for these sites. The IAF-labeled Ca2+-ATPase was completely proteolyzed with trypsin, followed by centrifugation to remove (unlabeled) membrane-associated portions. The soluble IAF-labeled tryptic peptides were purified by size-exclusion and reverse-phase high-performance liquid chromatography. Two IAF-peptides resulted. The major (4.1 nmol of IAF/mg of starting protein) and minor (1.9 nmol/mg) IAF-peptides were sequenced and were identified, respectively, as Ala673-IAF-Cys674-Cys675-Phe676-Ala677+ ++-Arg678 and as Glu668-Ala669-IAF-Cys670-Arg671. A model is proposed to explain the selectivity of IAF for Cys670 and Cys674 of the approximately 14 surface sulfhydryls of the Ca2+-ATPase. The labeling region, Arg667 through Arg678, has been predicted to be alpha-helical; Cys670 and Cys674 would be adjacent in the helix and imbedded in an Arg cluster. The Arg residues would both attract the anionic IAF and enhance sulfhydryl reactivities by lowering their pK values.
Bishop
J E
JE
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Squier
T C
TC
Bigelow
D J
DJ
Inesi
G
G
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D005452:Fluoresceins; D005456:Fluorescent Dyes; C050046:6-iodoacetamidofluorescein; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D003545:Cysteine