Interaction of unsaturated fatty acids with the red blood cell Ca(2+)-ATPase. Studies with a novel photoactivatable probe.
Unsaturated fatty acids, such as oleic acid, increase both the affinity for Ca2+ and the maximum effect of the Ca(2+)-ATPase of red blood cells [Niggli et al. (1981) J. Biol. Chem. 256, 8588-8592]. With the aim of examining the structural and kinetic details of the interaction between unsaturated fatty acids and 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 Ca(2+)-ATPase; (ii) in the absence of calmodulin, AS86 shows a biphasic behavior; i.e., at low concentrations it increases the affinity for Ca2+ and the maximum velocity of the enzyme, while at higher concentrations it decreases the maximum velocity; (iii) in the presence of calmodulin, AS86 increases slightly the affinity for Ca2+ and decreases the maximum velocity of the Ca2+ pump; and (iv) AS86 inhibits the activity of the enzyme devoid of its calmodulin-binding domain after proteolysis. When the reagent is covalently bound to the native enzyme, and then activated by calmodulin, increasing amounts of AS86 decrease the maximum velocity along a hyperbolic curve without modifying the apparent affinity for Ca2+. These results could be explained by the eventual existence of two different kind of sites recognizing the reagent: one influencing the affinity for Ca2+ and the other inhibitory of the calmodulin effects. When covalently bound, AS86 exerts its inhibitory effects upon the enzyme lacking the calmodulin-binding domain, 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 125I-AS86. Both the inhibitory effect on the calmodulin-dependent enzymic activity after covalent binding of AS86 and the photoadduct formation between the enzyme and 125I-AS86 are impaired by the presence of oleic acid in a concentration-dependent fashion. Recognition of photoreactive fatty acid analogues by the purified enzyme could be useful to provide further insight on the location of the interacting sites.
Rossi
J P
JP
Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
Delfino
J M
JM
Caride
A J
AJ
Fernández
H N
HN
D000328:Adult; D000345:Affinity Labels; D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004910:Erythrocyte Membrane / Q000201:enzymology*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008968:Molecular Conformation; D010777:Photochemistry; D012459:Salicylates / Q000138:chemical synthesis / Q000737:chemistry
C092735:8-(5'-azido-O-hexanoylsalicylamido)octanoic acid; D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D005231:Fatty Acids, Unsaturated; D012459:Salicylates; D000252:Calcium-Transporting ATPases