Metal-ATP complexes as substrates and free metal ions as activators of the red cell calcium pump

The plasma membrane calcium pump in most mammalian cells is the basic mechanism for assuring a low cytoplasmic calcium concentration. In inside-out human red cell membrane vesicles /IOVs/ the substrate and metal specificity as well as the intracellular protein /calmodulin/ regulation of the ATP-dependent active calcium transport can be investigated $\text{in}$$\text{situ}$. In this paper we demonstrate that Me²⁺. ATP^4? /in the following MeATP/ complexes, including MgATP, MnATP, CoATP, FeATP, and NiATP, can serve as substrates for the calcium pump in IOVs. Calcium pumping is activated by the above metals, while Sr, Ba, Cu, Cd ions or the trivalent cations are ineffective in this respect. Calmodulin-stimulation of the calcium transport is present independent of the metal ions used for the activation of the pump. Based on kinetic studies we suggest that divalent metal ions interact with the red cell calcium pump at four different sites: 1./ MeATP complex is the true substrate of the pump; 2./ Ca or Sr ions activate the system by binding to the transport site/s/ and other metal ions competitively inhibit this binding; 3./ the presence of free divalent metal ions /Mg, Mn, Co, Fe, or Ni, but not Ca, Sr, Ba/ is required for activating calcium translocation; 4./ interaction with a Ca — calmodulin complex specifically stimulates calcium pumping.