Alterations in phospholipid-dependent (Na+ +K+)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2+.
The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002784:Cholesterol / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology*; D008861:Microsomes / Q000201:enzymology; D010714:Phosphatidylethanolamines / Q000494:pharmacology*; D010718:Phosphatidylserines / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology; D011817:Rabbits; D012964:Sodium / Q000494:pharmacology; D013010:Sonication; D013696:Temperature; D013816:Thermodynamics
D008081:Liposomes; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D000255:Adenosine Triphosphate; D002784:Cholesterol; D012964:Sodium; D000251:Adenosine Triphosphatases; D008274:Magnesium; D011188:Potassium