Reactive Sulfhydryl Groups of Sarcoplasmic Reticulum ATPase. II. Site of Labeling with lodoacetamide and Its Fluorescent Derivative

Abstract

Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) aminonaphthalene-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 μM [¹⁴C] IAA at pH 7.0 and 30°C. One mole of IAA per mole of ATPase was bound in 6h 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 μM IAEDANS for 5 h, at pH 7.0 and 30°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.