Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. II. Site of labeling with iodoacetamide and its fluorescent derivative.
Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) amino-naphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+,Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 microM [14C]IAA at pH 7.0 and 30 degrees C. One mole of IAA per mole of ATPase was bound in 6 h without affecting the Ca2+-transport activity. [14C]IAA-labeled SR membranes were cleaved with BrCN, and 14C-labeled peptide fragments were separated by Sephadex LH-60 chromatography and then digested further with trypsin. A radioactive peptide (Ala-Cys 674-Cys-Phe-Ala-Arg) was purified by Sephadex LH-20 chromatography and C18 reversed phase HPLC (Cys denotes the [14C]IAA-binding site). IAEDANS-labeling was carried out by reacting SR membranes with 50 microM IAEDANS for 5 h, at pH 7.0 and 30 degrees C. A fluorescent peptide was successfully purified by the same procedures as for the IAA-labeled peptide, and the amino acid sequence analysis of this peptide revealed that the IAEDANS labeling site was identical with the IAA binding site.
D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002845:Chromatography / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism*; D007460:Iodoacetamide / Q000378:metabolism*; D007461:Iodoacetates / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D005456:Fluorescent Dyes; D007461:Iodoacetates; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; C006421:1,5-I-AEDANS; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D007460:Iodoacetamide