Interaction of Unsaturated Fatty Acids with the Red Blood Cell Ca2+-ATPase. Studies with a Novel Photoactivatable Probe

43. ROSSI J.P.F.C., CARIDE, A.J., DELFINO, J.M. AND FERNÁNDEZ, H.N.

BIOCHEMISTRY

AMER CHEMICAL SOC

Lugar: Washington; Año: 1995 vol. 34 p. 3802 - 3812

0006-2960

ABSTRACT: Unsaturated fatty acids, such as oleic acid, increase both the affinity for Ca2+ and the maximumeffect of the Ca2+-ATPase of red blood cells [Niggli et al. (1981) J. Biol. Chem. 256, 8588-85921. Withthe aim of examining the structural and kinetic details of the interaction between unsaturated fatty acidsand the enzyme, we designed and synthesized 8-(5?-azido-O-hexanoylsalicylamido)octanoic acid (AS86),a photoactivatable analogue of unsaturated fatty acids. AS86, interacting noncovalently with the enzyme,shares with oleic acid the following properties: (i) it binds reversibly to the plasma membrane Ca2+-ATPase; (ii) in the absence of calmodulin, AS86 shows a biphasic behavior; Le., at low concentrations itincreases the affinity for Ca2+ and the maximum velocity of the enzyme, while at higher concentrationsit decreases the maximum velocity; (iii) in the presence of calmodulin, AS86 increases slightly the affinityfor Ca2+ and decreases the maximum velocity of the Ca2+ pump; and (iv) AS86 inhibits the activity ofthe enzyme devoid of its calmodulin-binding domain after proteolysis. When the reagent is covalentlybound to the native enzyme, and then activated by calmodulin, increasing amounts of AS86 decrease themaximum velocity along a hyperbolic curve without modifying the apparent affinity for Ca2+. Theseresults could be explained by the eventual existence of two different kind of sites recognizing thereagent: one influencing the affinity for Ca2+ and the other inhibitory of the calmodulin effects. Whencovalently bound, AS86 exerts its inhibitory effects upon the enzyme lacking the calmodulin-bindingdomain, thus reflecting that this action is promoted by interaction with a site lying outside this region.The purified enzyme is susceptible to be tagged with 1251-AS86. Both the inhibitory effect on thecalmodulin-dependent enzymic activity after covalent binding of AS86 and the photoadduct formationbetween the enzyme and 1251-AS86a re impaired by the presence of oleic acid in a concentration-dependentfashion. Recognition of photoreactive fatty acid analogues by the purified enzyme could be useful toprovide further insight on the location of the interacting sites.Sarkadi et al. (1982) presented evidence indicating thatphospholipase A2 digestion products, namely, lysophosphatidylcholineand oleic acid, increase the rate of calcium pumpactivity in calmodulin-depleted inside-out vesicles from redblood cells. A similar effect of unsaturated fatty acids andacidic phospholipids upon the purified enzyme was reportedby Niggli et al. (1981b). However, Davis et al. (1987), atvariance with the earlier results, found an inhibitory responsefor oleic acid and the absence of any effect for otherunsaturated fatty acids, including linolenic and arachidonicacids. These observations are particularly significant inconnection with the widespread occurrence of agoniststimulatedphosphatidylcholine breakdown, associated withactivation of phospholipase A2 and release of unsaturatedfatty acids (Exton, 1990).On the basis of the response to acidic phospholipids ofCa2+ pump fragments resulting from limited trypsin proteolysis,Enyedi et al. (1987) and Zvaritch et al. (1990)proposed a