Journal of Cellular Biochemistry 46:284-290 (1 991 zy ) zy Association of Vanadate-Sensitive Mg2'-ATPase and Shape Change in Intact Red Blood Cells Y.-H. Xu, zyxwvutsrq Z.-Y. Lu, A.D. Conigrave, M.E. Auland, and B.D. Roufogalis Departments of Biochemistry (Y.-H.X., Z.-Y.Lu, A.D.C.) and Pharmacy (M.E.A., B.D.R), University of Sydney, Sydney, New South Wales 2006, Australia Abstract zyxwvutsrqpo Intact human erythrocytes, initially depleted of Mg" by EDTA incubation in the presence of A23187, exhibit M$+-dependentphosphate production of around 1.5 mmol per liter cells. h, half-maximally activated at around 0.4 mM added free M$'. This appears to correspond to Mg2'-stimulated adenosine triphosphatase (Mg2'-ATPase) activity found in isolated membranes, which is known to have a similar activity and affinity for Mg2+. Vanadate (up to 100 pM) inhibited Mg2+-dependent phosphate production and ATP breakdown in intact cells. Over a similar concentration range vanadate (3-1 00 PM) transformed intact cells from normal discocytes to echinocytes within 4-8 h at 37"C, and more rapidly in Mg2+-depleted cells. The rate of Ca2+-induced echinocytosis was also enhanced in Mg'-depleted cells. These results support previous studies in erythrocyte ghosts suggesting that vanadate-induced shape change is associated with inhibition of M&'-ATPase activity localized in the plasma membrane of the red blood cell. Key words: erythrocytes, magnesium, echinocyte, calcium, plasma membrane Human red blood cells undergo both ATP- dependent and ATP-independent transitions from smooth biconcave (discocytic) forms to crenated (echinocytic) states [ 13. Despite a con- siderable amount of detailed study over a num- ber of years, the exact mechanisms of red blood cell shape regulation remain uncertain. The bi- layer-couple hypothesis [2,31 proposes that cell shape is a function of the relative areas of the inner and outer leaflets of the membrane, such that anionic amphipaths, which accumulate mainly in the outer leaflet, cause echinocytosis by expanding the outer leaflet, whereas cationic amphipaths, which accumulate in the inner leaflet, produce cup shapes (stomatocytosis) by increasing the inner leaflet area. This hypothe- sis could also account for the ATP-dependent control of red cell shape [4], since lipid phospho- rylation by ATP could expand the inner leaflet by increasing its mass and negative charge den- sity [51. Dephosphorylation of membrane inosi- Received September 4,1990; accepted March 18,1991. Y.-H. Xu and zyxwvutsrqpo 2.-Y. Lu's present address is Department of Medical Biochemistry and Genetics, Texas A & M Univer- sity, College of Medicine, Texas. Address reprint requests to B.D. Roufogalis, Department of Pharmacy, University of Sydney, NSW 2006, Australia. to1 phospholipids during ATP-depletion may have the opposite effect and cause echinocytosis [61. It has also been proposed that phosphoryla- tion might be required for expansion of the cytoskeletal spectrin-actin network at the inner membrane surface [ 71, but echinocyte formation was subsequently shown to precede spectrin de- phosphorylation [8]. Furthermore, ATP-depen- dent phosphorylation of spectrin could be disso- ciated from ATP-dependent shape change, since smoothing of echinocytes in red cell ghosts de- pleted of spectrin kinase was unaltered and both spectrin and phosphoinositide phosphorylation were unaffected by vanadate at concentrations which blocked shape changes in ghosts [91. Echinocyte formation by ATP-depletion or Ca2' loading of red blood cells has also been corre- lated with phosphoinositide [6] or phosphatidic acid [lo] breakdown, resulting in shrinkage of the inner leaflet 1161. Other models of shape change have also been proposed. ATP-dependent translocation of ami- nophospholipids from the outer to the inner leaflet by the phospholipid translocase may serve to maintain the balance between the area of the bilayer halves [11,121. In another proposal a direct effect of ATP binding to the cytoskeleton was postulated from results showing that ATP z 0 1991 Wiley-Liss, Inc.