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An Iodoacetamide Spin-Label Selectively Labels a Cysteine Side Chain in an Occluded Site on the Sarcoplasmic Reticulum Ca(2+)-ATPase

An Iodoacetamide Spin-Label Selectively Labels a Cysteine Side Chain in an Occluded Site on the Sarcoplasmic Reticulum Ca(2+)-ATPase

A Wawrzynow, J H Collins, C Coan

Biochemistry. 1993 Oct 12;32(40):10803-11.

PMID: 8399229

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP73264127	6-(Iodoacetamido)fluorescein		73264-12-7	Price

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