Fluorescence Spectroscopy in the Study of Sarcoplasmic Reticulum Calcium-ATPase

Abstract

Sarcoplasmic reticulum is an intracellular membrane system that serves as a model for active transport of Ca²⁺ and coupled enzyme catalysis in biological membranes. The isolation of vesicular fragments of sarcoplasmic reticulum membranes from muscle homogenates and the demonstration that they were capable of maintaining calcium transport at the expense of the hydrolysis of ATP (Ebashi and Lipman, 1962; Hasselbach and Makinose, 1961) has led the way to extensive characterization of the kinetics and mechanism of calcium transport and energy transduction in this system. The work with sarcoplasmic reticulum has been extensively reviewed (de Meis, 1980; Ikemoto, 1982; Inesi, 1985). The sarcoplasmic reticulum membranes have a highly specific protein composition, consisting of a Ca²⁺- dependent ATPase of 115,000 daltons per polypeptide chain. The ATPase accounts for 80%-90% of the total protein, and its localization within the membrane has been characterized both by X-ray diffraction from oriented multilayers (Dupont et al, 1973; Herbette et al., 1977) and by electron microscopy (Deamer and Baskin, 1969; Hasselbach and Elfvin, 1967).These studies have demonstrated that the ATPase is densely spaced within the plane of the membrane and that the ATPase chain has an amphiphylic character, with a hydrophobic portion inserted into the membrane bilayer and a hydrophylic portion protruding from the cytoplasmic surface of the membrane into the aqueous phase. More recently, the amino acid sequence of the ATPase has been established by MacLennan et al (1985) and a computer-simulated structural model has been proposed from the primary sequence.