Analysis of microsomal calcium sequestration by steady state isotope exchange. Enzyme kinetics and role of membrane permeability.

ATP-dependent calcium sequestration by rat liver microsomes has been analyzed by steady state isotope exchange. Liver microsomes display high affinity for Ca2+; the half-maximal concentration of free Ca2+ is 0.10 microM, and intravesicular steady state concentrations of 7-8 mM Ca2+ are achieved under optimal conditions. The uptake system displays multiphasic kinetics with respect to both Mg-ATP and free Mg2+, suggesting that microsomal preparations contain two distinct Ca2+-sequestering systems. Measurement of the kinetics of Ca2+ sequestration permits independent assessment of the activity of the calcium active transport system(s) and of the permeability of the membrane to Ca2+ backflux. Addition of ionophore A-23187 to microsomes renders them more permeable, and this is reflected in a more rapid equilibration of isotope. Conversely, low levels of free Ca2+ lead to a decreased rate of active transport, and this is reflected in a lower initial rate of isotope exchange. This system should be useful for investigating the mechanisms by which hormones, hepatotoxins, and other agents influence Ca2+ fluxes in cells.