Effect of phospholipid hydrolysis by phospholipase A 2 on the kinetics of antagonist binding to cardiac muscarinic receptors

• Published in: Biochemical pharmacology Vol. 48; no. 6; pp. 1289 - 1296
• Main Authors: Rauch, Bernhard, Niroomand, Feraydoon, Messineo, Frank C., Weis, Angelika, Kübler, Wolfgang, Hasselbach, Wilhelm
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 1994
• Subjects: antagonist binding; binding kinetics; cardiac muscarinic receptors; phospholipase A 2; quinuclidinyl benzilate; sarcolemma; cardiac muscarinic receptors; quinuclidinyl benzilate; antagonist binding; R 0; QNB; phospholipase A 2; binding kinetics; v 0; K d; R; R-QNB; B max; V max; K m; k 2 and k −2; PLA 2; K 1; sarcolemma
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/0006-2952(94)90167-8

Activation of phospholipases during prolonged myocardial ischemia could contribute to the functional derangement of myocardial cells by altering the phospholipid environment of a number of membrane bound proteins including receptors. The present study examined the kinetics of muscarinic receptor antagonist [ 3H] quinuclidinyl benzilate binding ([ 3H]QNB) to muscarinic receptors of highly purified sarcolemmal membranes under control conditions and after treatment with phospholipase A 2 (PLA 2; EC 3.1.1.4.). Initial binding rates of QNB exhibited saturation kinetics, when plotted against the ligand concentration in control and PLA 2 treated sarcolemmal membranes. This kinetic behaviour of QNB-binding is consistent with at least a two step binding mechanism. According to this two step binding hypothesis an unstable intermediate receptor-QNB complex (R*QNB) forms rapidly, and this form undergoes a slow conversion to the high affinity ligand-receptor complex R-QNB. The Michaelis constant K m of R-QNB formation was 1.8 nM, whereas the dissociation constant K d obtained from equilibrium measurements was 0.062 nM. After 5 min exposure of sarcolemmal membranes to PLA 2QNB binding capacity ( B max) was reduced by 62%, and the affinity of the remaining receptor sites was decreased by 47% ( K d = 0.116 nM). This PLA 2-induced increase of K d was accompanied by a corresponding increase of K m , whereas the rate constants k 2 and k −2 of the hypothetical slow conversion step (second reaction step) remained unchanged. These results suggest that binding of QNB to cardiac muscarinic receptors induces a transition in the receptor-ligand configuration, which is necessary for the formation of the final high affinity R-QNB complex. PLA 2-induced changes of the lipid environment result in the inability of a part of the receptor population to undergo this transition, thereby inhibiting high affinity QNB-binding.