A spin-label study of phosphatidylcholine exchange protein. Regulation of the activity by phosphatidylserine and calcium ion

Abstract

Exchange of phosphatidylcholine catalyzed by exchange protein has been studied by a new technique using spin-labeled phosphatidylcholine (PC∗). The exchange activity was assayed by the change in electron spin resonance (ESR) spectrum when PC∗ vesicles were incubated with unlabeled phospholipid vesicles. The method utilized decrease in the spin-spin exchange interaction and does not require separation of the donor and acceptor vesicles. 1. (1) The transfer rate, υ, from PC ∗ to phosphatidylcholine vesicles described by υ = k [PC ∗][protein] (1 + K[ PC]) for large excess of a acceptor vesicles where k is the rate constant for association of the protein with PC ∗ vesicles and K the binding constant of the protein with phosphatidylcholine vesicles. The transfer rate at 23°C was 3.2 μ mol · min −1 · ( protein) −1 · ( M PC ∗ ) −1 at [PC] = 14.7 mM, k = 136 mM −1 · min −1, and K = 79 M −1. 2. (2) The exchange activity was inhibited by addition of phosphatidylserine vesicles and also by mixed phosphatidylserine-phosphatidylcholine vesicles. The inhibition was abolished by Ca 2+ and Mg 2+. 3. (3) The ESR spectrum of PC ∗ complexed with exchange protein showed strong immobilization of the lipid alkyl chain, suggesting van der Waals-type binding of the acyl chain to the protein interior. The endogeneous PC ∗ molecule was readily exchanged with phosphatidylcholine but practically inexchangeable with phosphatidylserine in the vesicle membranes. 4. (4) A gel chromatographic analysis indicated weak interaction of the exchange protein with phosphatidylcholine vesicles but strong binding to phosphatidylserine vesicles. The inhibitory effect of the anionic lipid vesicles can be explained by their binding to the protein and incapability of exchange of the endogenous phosphatidylcholine with phosphatidylserine in the anionic membranes. The restoration by Ca 2+ and Mg 2+ may be due to binding of these cations to the anionic lipids, making the protein free from the vesicles. The divalent cations thus act regulatorily for the exchange reaction.