Chemical modification of the Ca(2+)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum: identification of sites labeled with aryl isothiocyanates and thiol-directed conformational probes.
The Ca(2+)-ATPase protein of rabbit skeletal muscle sarcoplasmic reticulum is a single polypeptide chain of 1001 amino-acid residues. Among these residues are 24 Cys, 9 of which have previously been shown to be accessible to one or more thiol-specific reagents. Many studies on the structure and function of this Ca(2+)-ATPase have made use of sulfhydryl-directed, conformationally-sensitive probes, but the labeling sites for these probes have been directly identified in only a few cases, causing uncertainty in the interpretation of results. In the present work, we have investigated the Ca(2+)-ATPase labeling sites for three thiol-directed spectroscopic probes: fluorescein 5'-maleimide (Fmal), 4-dimethylaminophenyl-azo phenyl-4'-maleimide (DABmal), and 4-dimethylaminophenylazophenyl-4'-iodoacetamide (DABIA). Labeled Ca(2+)-ATPase was digested exhaustively with trypsin, and labeled peptides were purified and sequenced in order to identify the labeled Cys residues. Our results do not support the widely held assumptions that Cys-344 and Cys-364 are the most reactive residues with maleimide-based reagents, while Cys-670 and Cys-674 react most rapidly with iodoacetamide derivatives. We found instead that Fmal reacted most rapidly with Cys-471, followed by Cys-364, and more slowly with Cys-498, -525, -614 and -636. DABmal reacted most rapidly with Cys-364, followed by Cys-614, and more slowly with Cys-471, -498, -636 and -670. Cys-344 was not labeled by either Fmal or DABmal. DABIA reacted with the same six Cys residues, including Cys-670, as were labeled with DABmal, but in much lower yield. There was no evidence for labeling of Cys-674 with DABIA. The high reactivity of Fmal, but not the more hydrophobic DABmal, with Cys-471 is of interest because of previous studies suggesting that the accessibility of Cys-471 is influenced by ATP and that fluorescein derivatives bind to a hydrophobic pocket in the ATP binding site. Another derivative, fluorescein-5'-isothiocyanate (FITC), is thought to label the catalytic site of the Ca(2+)-ATPase and has been widely used as a conformational probe in structure-function studies on this and related proteins. We reinvestigated the chemical modification of the Ca(2+)-ATPase by FITC and 4-dimethyl-aminophenyl-4'-isothiocyanate (DABITC). Incorporation of stoichiometric amounts of FITC resulted in a nearly complete loss of ATPase activity. Labeling and inactivation of the Ca(2+)-ATPase by FITC did not occur in the presence of ATP. DABITC was less reactive than FITC, and did not inactivate the Ca(2+)-ATPase to any significant extent.(ABSTRACT TRUNCATED AT 400 WORDS)
Wawrzynów
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Department of Biological Chemistry, School of Medicine, University of Maryland, Baltimore 21201.
Collins
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate; D017879:Isothiocyanates; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry*; D010446:Peptide Fragments; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D014357:Trypsin; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D017879:Isothiocyanates; D015335:Molecular Probes; D010446:Peptide Fragments; C012926:4-(N,N-dimethylaminoazobenzene)-4'-isothiocyanate; D004124:p-Dimethylaminoazobenzene; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate