Partial purification and characterization of the human erythrocyte Mg²⁺ ‐ATPase A candidate aminophospholipid translocase

Abstract

A Mg2÷-ATPase-enriched fraction was obtained from solubilized human erythrocyte membranes by ammonium sulphate precipitation and anionexchange chromatography. The solubilized enzyme, of apparent molecular weight 120 kDa, requires phosphatidylserine to be fully active. Phosphatidylethanolamine but not other anionic phospholipids can only partially restore the activity. The Mg-ATPase has a low affinity for Mg2÷-ATP and is inhibited by fluoride, vanadate, vanadyl and calcium ions. From these characteristics, we infer that this Mg2+-ATPase is the same protein as the aminophospholipid translocase which regulates the membrane phospholipid transverse distribution in human erythrocytes by actively transporting aminophospholipids from the outer to the inner monolayer.

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function

“…Chromaffin granule ATPase II requires Mg 2ϩ for ATP hydrolysis (24), and the red blood cell membrane also contains a Mg 2ϩ -dependent ATPase that appears to be an APLT (25). We found that the addition of EDTA to chelate Mg 2ϩ inhibited the ATP-dependent translocation of NB-PS to the cytosolic leaflet (Fig.…”

“…(iii) The erythrocyte Mg 2ϩ -ATPase does not seem to pump Mg 2ϩ , H ϩ , or other ions tested across the erythrocyte plasma membrane (30). (iv) PS stimulates ATP hydrolysis by ATPase II and the erythrocyte Mg 2ϩ -ATPase, suggesting that it is a substrate of these enzymes (25,29). (v) Phylogenetic analysis of the Drs2p family of P-type ATPases indicates a substantial divergence from ion and heavy-metal transporters (31).…”

The role of phosphatidylserine in recognition of apoptotic cells by phagocytes

“…A membrane protein termed the aminophospholipid translocase transfers PS and to some extent, PE, from the outer leaflet to the inner leaflet (Devaux, 1991;Zachowski, 1993;Williamson and Schlegel, 1994). This protein appears to be a Mg 2+ -ATPase which is inhibited by Ca 2+ at concentrations achieved in many apoptotic cells (Daleke and Huestis, 1985;Zachowski et al, 1986;Bitbol et al, 1987;Bevers et al, 1989;Morrot et al, 1990;Comfurius et al, 1990;Auland et al, 1994). Tang and coworkers recently cloned a novel P-type ATPase with aminophospholipid translocase activity (Tang et al, 1996).…”

Transbilayer Phosphatidylethanolamine Movements in the Yeast Plasma Membrane

“…Sodium orthovanadate is an inhibitor of most of the nncleotide-dependent processes and is thought to exert its effect by mimicking the phosphate group at the nucleotide-binding site [28]. It has been used to inhibit the activity of the aminophospholipid translocase in partially purified membrane fractions [29] and intact cells [6,301, and is hence a good choice for the present studies. Yeast cells were incubated with different concentrations of sodium orthovanadate as described in Materials and Methods.…”