Distances between functional sites of the Ca²⁺+ Mg²⁺‐ATPase from sarcoplasmic reticulum using Co²⁺ as a spectroscopic ruler

Abstract

Cobalt ion inhibits the Ca2+ + Mg2 +-ATPase activity of sealed sarcoplasmic reticulum vesicles, of solubilized membranes and of the purified enzyme. To use Co2+ appropriately as a spectroscopic ruler to map functional sites of the Ca2+ + Mg2+-ATPase, we have carried out studies to obtain the kinetic parameters needed to define the experimental conditions to conduct the fluorimetric studies.1. The apparent Ko.5 values of inhibition of this ATPase are 1.4 mM, 4.8 mM and 9.5 mM total Co2+ at pH 8.0, 7.0 and 6.0, respectively. The inhibition by Co2+ is likely to be due to free Co2+ binding to the enzyme. Millimolar Ca2 from which we obtain a distance of 1.1 -1.9 nm between Co2+ and fluorescein located at neighbouring catalytic sites. Co2+. ATP can be used as a substrate by this enzyme with V,,, of 2.4 f 0.2 pmol ATP hydrolyzed min- Co2+ partially quenches, about 10The sarcoplasmic reticulum Ca2+ + Mg2 +-ATPase uses Mg2+ . ATP as its physiological substrate and couples ATP hydrolysis to the active transport of Ca" across this membrane [l]. Binding of two Ca2+ ions to high-affinity binding sites/ATPase molecule is step that precedes Ca2+ transport to the luminal space of sarcoplasmic reticulum [l, 21. To understand better the Ca2+ transport process coupled to ATP hydrolysis requires complementary experimental information obtained by using kinetic and structural studies of this enzyme. In this regard, the location of Ca2+ and ATP binding sites relative to each other is an important issue; there is a large range of values for the distance between these sites on ATPase reported by different groups of investigators, i. e. 1 -6 nm [3 -71. Forster energy transfer between different donor/acceptor pairs of fluorescent targets selectively bound to these sites has been used to obtain these estimations. On the other hand, Xray diffraction studies [8, 91 and image processing of electron microscopy data [lo, 111 have provided evidence for an overall shape of an ATPase unit protruding about 4.0 -6.0 nm from the lipid/water interface and having a cross-sectional diameter of the cytosolic lobule of approximately 8.0-9.0 nm in its dimeric form, which is probably the predominant state in

Decavanadate induces mitochondrial membrane depolarization and inhibits oxygen consumption

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Fluorescence energy transfer as a tool to locate functional sites in membrane proteins

“…In practice, it is usually the uncertainty of the value of the orientation factor, K', that more severely limits the precision of distance measurements between a single donor/acceptor pair. When the shape of the electron-density map of the acceptor is close to a spherical geometry (for example, for cations such as Co'+ [9]), the random orientation assumption is well justified. I Iowever, most fluorescent dyes are molecules of close to planar geometry.…”

Mechanisms of cobalt(II) uptake into V79 Chinese hamster cells

“…active transport systems. Since Co 2+ could interact with the Ca 2+ binding site of a Ca2+/MgZ+ATPase as observed for the sarcoplasmic reticulum enzyme (Cuenda et al 1990), a Co2+ transport via exchange for Ca 2+ would be conceivable. Alternatively, ATP could bind Co(II), and this Co(II)-ATP-complex could substitute for MgZ+-ATP as co-substrate (Yamada and Ikemoto 1980).…”