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.
Wawrzynow
A
A
Department of Biological Chemistry, School of Medicine, Maryland Biotechnology Institute, University of Maryland, Baltimore 21201.
Collins
J H
JH
Coan
C
C
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