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A soluble delipidized (Na + K)-stimulated ATPase was obtained from rabbit kidney outer medulla by the use of sodium deoxycholate. The activity of this enzyme was stimulated 20-fold to an activity of 100 to 120 µmoles of Pi per mg of protein per hour by either phosphatidylserine or phosphatidylglycerol. The effect of the fluidity of the hydrocarbon region of the phospholipid bilayers on this re-activation of (Na + K)-ATPase was systematically examined. Fatty acyl chains of known length and varying saturation were substituted on the phosphatidylglycerol moiety and the influence of temperature on the degree of re-activation was investigated. Discontinuities in Arrhenius plots of (Na + K)-ATPase activity were found and compared with the gel-to-liquid-crystalline transitions of these substituted phosphatidylglycerols as measured in a differential scanning calorimeter. The temperature at which the Arrhenius plot discontinuities occurred were from 1° to 8° lower than the initial rise of the main endothermic peak for the series: dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylglycerols. Dioleoyl-phosphatidylglycerol which does not undergo a phase transition within the experimental temperature range was found to activate at much lower concentrations than the saturated phosphatidylglycerols, and the Arrhenius plot did not show discontinuities. In contrast, bovine brain phosphatidylserine with a heterogeneous fatty acyl chain composition showed an Arrhenius plot discontinuity which was 2° higher than the midpoint of the broad endothermic peak. The relation between the discontinuities in the Arrhenius plots of the enzyme activity and the lipid phase transitions is discussed. It is suggested that some of the discontinuities represent the completion of melting (upper limit for a broad endothermic transition), whereas others represent the beginning of melting (lower limit). For completely liquid membranes, the slopes of the Arrhenius plots are parallel and give an activation energy of approximately 15 Cal per mole. The influence of membrane fluidity was also studied by examining the effects of cholesterol at constant temperature (37°). In agreement with the above findings, cholesterol, which is known to reduce the fluidity of phospholipid fatty acyl chains, inhibits phospholipid-stimulated (Na + K)-ATPase activity. The inhibition is complete, however, only with saturated phospholipids. Partial inhibition is obtained with unsaturated phospholipids. These results are discussed in relation to phase transitions in phospholipid bilayers and biomembranes and the activity of membrane-bound enzymes. A role of cholesterol in controlling the activity of membrane-bound enzymes is suggested. Implications for protein-lipid interactions in membranes are also considered.
Kimelberg et al. (Fri,) studied this question.