Comp. Biochem. Physiol. Vol. 82B, No. 1, pp. 117-122, 1985 0305-0491/85 $3.004-0.00 Printed in Great Britain © 1985PergamonPress Ltd COMPARISON OF THE MEMBRANE-BOUND (Ca2+ + MgZ+)-ATPase IN ERYTHROCYTE GHOSTS FROM SOME MAMMALIAN SPECIES CLEMENT O. BEWAJI,* OLUFUNSO O. OLORUNSOGOand ENITAN A. BABABUNMI'~" Biomembrane Research Laboratories, Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan, Nigeria (Received 20 February 1985) Abstraet--I. The properties of the membrane-bound calcium-pumping protein, the (Ca2+ +Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) were compared in erythrocyte ghosts isolated from five mammalian species--human (Homo sapiens), bovine (Bos taurus), porcine (Sus scrofa melitensis), ovine (Ovis aries crassicandus) and caprine (Capra hircus syriaea). 2. The specific activity of the enzyme in porcine erythrocytes is one order of magnitude higher than in the other species. It was also stimulated to various extents by the regulator protein, calmodulin, and by phosphatidylinositol in all the species. 3. Analysis of membrane proteins revealed a number of differences which seem to suggest that the molecular architecture of the red cell membrane influences the activity of the enzyme. INTRODUCTION Increasing interest in the role of membrane proteins has led to efforts towards fractionating and charac- terizing the proteins of the erythrocyte membrane. The proteins of the human erythrocyte membrane have been analyzed and characterized by several investigators (Fairbanks et al., 1971; Tanner and Boxer, 1972; Steck, 1974; Tomita and Marchesi, 1975). It has become clear in recent years that the inter- actions of lipids with enzymic proteins in biological membranes play an important role in the activity of these enzymes (Fourcans and Jain, 1974; Ronner et al., 1977). One such protein is the (Ca2+ + MgZ+)-ATPase which is now generally ac- cepted as the enzyme responsible for the active extrusion of Ca 2+ across the plasma membrane of erythrocytes (Schatzmann, 1975; Roufogalis, 1979; Carafoli et al., 1980). The successful physiological functioning of erythrocytes depends in part on their ability to maintain the intracellular concentration of Ca z+ below 1/~M. In 1973, Bond and Clough reported the presence, in human erythrocytes, of a protein activator of the (Ca 2+ + Mg2+)-ATPase. Jarrett and Penniston (1978) purified this protein which is now called calmodulin. However, recent studies have shown that calmodulin is not unique as an activator of the (Ca2+ + Mg2+)-ATPase of erythrocyte membranes. The activity of the enzyme can be considerably increased, in the absence of calmodulin, after per- turbations produced by a variety of acidic phos- pholipids or unsaturated fatty acids, phospholipase *Permanent address: Department of Biochemistry, Univer- sity of Ilorin, Ilorin, Nigeria. tPresent address: Department of Biochemistry, Chelsea College, University of London, Manressa Road, London SW3, UK. A 2 and mild proteolysis with trypsin (Taverna and Hanahan, 1980; A1-Jobore and Roufogalis, 1981; Niggli et aL, 1979). In the present paper, we have analyzed the polypeptide components of ghost membranes isolated from human, bovine, porcine, ovine and caprine erythrocytes. We have also compared the kinetic properties of the membrane-bound (Ca 2÷ + Mg2+)-ATPase especially with respect to its interaction with calmodulin, phosphatidylinositol and vanadate. Data concerning comparative bio- chemical characteristics of this important calcium- pumping ATPase are rather sparse. MATERIALS AND METHODS All the reagents used were of the highest purity available. ATP (vanadium-free), HEPES and fatty acid-free bovine serum albumin were obtained from Sigma Chemical Co., DEAE-cellulose (DE 52), sodium dodecyl sulphate, EDTA and EGTA were from Fluka AG, Switzerland, Sephadex G-100 (superfine) was from Pharmacia, Uppsala, Sweden. Phosphatidylinositol was obtained from Koch-Light Lab- oratories, Bucks, England, and sodium orthovanadate from ICN Pharmaceuticals, Inc., Plainview, NY. All other salts were analytical reagent grade. Preparation of erythrocyte ghost membranes Haemoglobin-free ghost membranes deficient in cal- modulin were prepared by the procedure of Niggli et al. (1979), with slight modifications. Recently outdated human blood was obtained from the Blood Bank of the University College Hospital, Ibadan, Nigeria. Fresh blood was also obtained from cow, pig, sheep and goat at a local slaughter- house. The blood from the various species was collected in acid citrate dextrose buffer, stored at 4°C and processed within 2 days. All the steps of the membrane preparation were carried out at 4°C unless otherwise stated. The whole blood was centrifuged at 5800g for 10rain. The plasma and "buffy" layers were removed by aspiration to obtain packed erythrocytes. The erythrocytes were washed twice in 5 vol. of 130 mM KCI, 20 mM Tris, pH 7.4. The cells were then haemolysed in 5 vol. of 1mM EDTA, 117