(Iodoacetamido)fluorescein labels a pair of proximal cysteines on the calcium ATPase of sarcoplasmic reticulum

Abstract

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.

p53 at the endoplasmic reticulum regulates apoptosis in a Ca 2+ -dependent manner

“…6), thus modulating SERCA activity. Indeed, prooxidative modification of SERCA decreased its activity (29), and a critical cysteine in the SERCA sequence (41) that regulates this posttranslational modification is accessible from the cytosolic compartment (42), where p53 is localized.…”

High glucose oxidizes SERCA cysteine-674 and prevents inhibition by nitric oxide of smooth muscle cell migration

“…Decreased biotinylated iodoacetamide binding to SERCA in smooth muscle cells exposed to high glucose indicates that cysteine-674, which accounts for more than 75% of IAM binding, [8,19] is oxidized. An increase in oxidants associated with high glucose is also indicated by the fact that treatment with the antioxidant, Tempol, prevented the decrease in b-IAM binding.…”

Regulation of Cell Physiology and Pathology by Protein S-Glutathionylation: Lessons Learned from the Cardiovascular System

“…In addition to myofibrils and the cytoskeletal proteins modulated by thiol exchange, elevated RNOS production at the ER and the plasma cell membrane can modulate cellular contraction by redox signaling and oxidative stress. In the ER/SR, RYR 2 (138,154,155,175) and SERCA2 (2,21,46,92,143,168) are exquisitely sensitive to thiol modifications leading to functional changes. In addition, plasma membrane bound proteins can also theoretically be regulated by oxidation due to the presence of oxidant enzymes such as NOXs.…”