Lipid exchange between mixed micelles of phospholipid and triton X-100

If phospholipase catalyzed hydrolysis of phospholipid dissolved in a deterg ent mixed micelle is limited to the phospholipid carried by a single micell e, then hydrolysis ceases upon exhaustion of that pool. However, if the rat e of phospholipid exchange between micelles exceeds the catalytic rate then all of the phospholipid is available For hydrolysis. To determine phosphol ipid availability we studied the exchange of 1, 2-dioleoyl-sn-glycero-3-pho sphocholine between mixed micelles of phospholipid and non-ionic Triton det ergents by both stopped-flow fluorescence-recover and nuclear magnetic reso nance-relaxation techniques. Stopped-flow analysis was performed by combini ng mixed micelles of Triton and phospholipid with mixed micelles that conta ined the fluorescent phospholipid 1-palmitoyl-2-(12-[{7-nitro-2-1,3-benzoxa diazo-4-yl}amino]dodecanoyl)-sn-glycero-3-phosphocholine (P-2-NBD-PC). The concentration dependence of fluorescence recovery suggested a second-order exchange mechanism that was saturable. The true second-order rate constant depends on the specific mechanism for exchange, which was not determined in this study, but the rate constant will be on the order of 10(6) to 10(7) M (-1)s(-1). Incorporation of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcho line into micelles increased the rate of proton relaxation and gave a limit ing relaxation time of 1.3 ms. The results demonstrate that phospholipid ex change was rapid and that the phospholipid content of a single micelle did not limit the rate of phospholipid hydrolysis by phospholipases. (C) 1999 E lsevier Science B.V. All rights reserved.