i J. Bififlit-m.. Vol. 12. pp. 295 lo 299 Porgamon Prcs:. Ltd 1980. Printcd in Gréai Brilain 0020-71 IX,HO/070I-0295S02.00/0 OPERATION OF THE RENAL Na + -K + -PUMP IN ARTIFICIAL MEMBRANES BEATRICE M. ANNER Department of Pharmacology, Ecole de Médecine CH-1211 Geneva 4, Switzerland Abstract—I. Na + -K + -ATPase was purified from thé outer medulla of rabbit kidneys and incorporated into artificial vesicular membranes (liposomes). Within 60 min of thé addition of ATP to thé liposomes, thé intravesicular Na"1" concentration rosé from 20 to about 65 m M whereas thé K + concentration dropped from 50 to about 30 mM. 2. Alkylation of SH groups on thé reconstituted pump protein with N-ethylmaleimide inactivated 30% of thé ATP-driven Na*-transport. K+ -transport appeared unaffected. 3. Occupation of thé phosphorylation site by thé natriuretic vanadate abolished thé total Na*- transport capacity of thé reconstituted Na + -K + -pump. 4. From thé results described it appears that thé in vitro System may be a useful model for simulating effects of extrinsic agents on thé rénal Na + -K + -pump. INTRODUCTION It is likely that thé rénal Na+-K + -pump (Na + -K+ - ATPase) is responsible for most, if not ail, thé Na + reabsorption in thé thick ascending loop of Henle (Schmidt & Dubach, 1969; Epstein & Silva, 1974; J0rgensen, 1976). When thé Na*-K+-ATPase from thé rénal outer medulla was purified and incorporated into phosphatidylcholine-Hposomes, a functional pump was obtained which performed coupled active Na + and K + transport and was inhibited by ouabain (Anner et ai, 1976, 1977; Goldin, 1977). Examination of thé purified Na +-K "^-transport System in artificial membranes led to thé characteriz- ation of possible transport pathways which may be cautiously extrapolated to thé in vivo situation (Anner 1980). In thé présent work, thé reconstituted Na + -K*- pump was chemically modified either by alkyfating SH groups with Af-ethylmaleimide or by blocking thé phosphorylation site with vanadate, and thé resulting altération of thé Na + and K + transport capacity was determined. It appeared that only a labile fraction of thé active Na+-transport was suppressed by thé N-ethylmaleimide treatment. In contrast, thé potent natriuretic vanadate (Balfour et ai, 1978) totally abol- ished Na + -transport. The results encourage thé use of thé in vitro System as a model for establishing transport patterns of thé rénal Na +-K+-pump. MATERIALS AND METHODS Préparation of soluble Na*-K Na + -K + -ATPase from thé outer medulla of rabbit kid- neys was purified to a spécifie activity of 800-1200 ^mol PjTng protein" 1 -hr' 1 according to thé angle rotor pro- cédure described by J0rgensen (1974). For solubilization, 1 mg Na + -K + -ATPase protein was suspended at 0°C in 500^1 of a 1% sodium cholate solution in buffer A (20 mM NaCl, 50 mM KC1, 50 mM choline choline chloride, 5 mM MgCl2, 30 mM imidazole, 1 mM L-cystein chloride, 1 mM EDTA, pH 7.1). The suspension was stirred for 60sec at 22CC and then centrifuged 15min at 100,000 g. The clear supernatant contained about 50% of thé initial Na +-K + - ATPase protein. The method of Lowry et ai (1951) was used for détermination of protein, except that 0.1% sodium dodecyl sulfate was présent. Incorporation of N a*-K*-AT Pose into liposomes Reconstitution was performed by a technique developed from previous procédures (Goldin & Tong, 1974; Hilden et ai, 1974; Anner et ai, 1977). A hexane solution containing 1 g per 10ml egg phosphatidylcholine (Sigma type III-E) was dried under a stream of N2 in a rotavapor. To 20 mg dried phospholipid 2.5ml of thé 1% cholate solution in buffer A were added and thé lipid was dissolved by rotai- ing at 22°C. For reconstitution, an aliquot of thé 1% cholate-Iipid solution was added to an aliquot of thé 1% cholate-enzyme solution (in ice) at a 1:1 ratio and thé mixture was dialysed for 90 hours at 5°C against a 1000-fold excess of buffer A. Electron micrographs revealed a homogenous popula- tion of 1000 À vesicles which contained Na + -K+ -pump particles spanning thé lipid bilayer (Skriver et al., in prép- aration). Transport measuremenis Aliquots of thé liposomes suspension were loaded with 22 Na and 86Rb and isotope équilibration was followed by passing 10/d samples over Sephadex-G-50 (médium) columns (1 x 20cm) and collecting thé eluted liposomes for scintillation counting. Active transport was initiated by adding 5 mM Tris-ATP to thé liposomes; this induced ac- cumulation of Na"^ in thé liposomes and extrusion of K* from thé liposomes. The ATP had been freed from vana- date by treatment with Dowex WX2 resin (Hudgins & Bond, 1977). RESULTS Transport pattern of thé rénal Na+-K+-pump in lipo- somes For thé experiments shown in Fig. 1, thé liposomes had been prepared in 20 mM NaCl and 50 mM KO, and were then equilibrated with 22Na and 86Rb (not shown). The rate constants for passive équilibration were 0.014min' 1 for K + and 0.013min-' for Na + , indicating that thé permeability of thé liposomes for Na + and K + is not much différent (Anner, 1980). 295