An iodoacetamide spin-label selectively labels a cysteine side chain in an occluded site on the sarcoplasmic reticulum Ca(2+)-ATPase.
Sarcoplasmic reticulum vesicles were labeled with [14C]iodoacetamide spin-label (ISL) under conditions where time courses of the reaction predicted that one amino acid residue would be preferentially labeled. Solubilized tryptic peptides were separated by high-performance liquid chromatography following extensive digestion, and amino acid sequences were determined for major and minor radio-labeled peptides. Only one radio-labeled residue, Cys-674 on the Ca(2+)-ATPase, could be identified. Extensive incubation with excess label increased nonspecific labeling, but did not produce detectable amounts of any other reactive side chain residue. Time courses of the iodoacetamide spin-label reaction were compared to those of 6-(iodoacetamido)fluorescein (IAF), and the ISL reaction was found to be more selective, in accordance with previous studies showing that IAF labeled both Cys-674 and Cys-670 [Bishop, J. E., Squire, T. C., Bigelow, D. J., & Inesi, G. (1988) Biochemistry 27, 5233-5240]. Titrations with spin-broadening reagents NiCl2 and Ni-EDTA showed Cys-674 to be in a region with very low solvent accessibility. These titrations also showed the ATPase to be distributed between two alternating conformations based on the accessibility of the label to NiCl2.
Department of Biological Chemistry, School of Medicine, Maryland Biotechnology Institute, University of Maryland, Baltimore 21201.
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Wawrzynow
J H
JH
Collins
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Coan
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002250:Carbon Radioisotopes; D003497:Cyclic N-Oxides / Q000138:chemical synthesis / Q000378:metabolism*; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013113:Spin Labels / Q000138:chemical synthesis; D014357:Trypsin
D002250:Carbon Radioisotopes; D003497:Cyclic N-Oxides; D010446:Peptide Fragments; D013113:Spin Labels; C003028:N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D003545:Cysteine