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TI-Pos321 T'u-Pos323 and CHOLESTEROL. ((A.C iismalanlr AvWDMR rxunnbP12^R1;| qjrAI bCrDTBTVWTTTI-MuIL-1 UKM AA"ANVn nTVAMT.4v I NAMAlb AAUIAini TI-Pos321 Ib-Pos322 Dithionite Quenching Rate Measurement of the Inside-Outside MEASUREMENT OF FLUORESCENCE SIGNAL OF A Membrane Bilayer Distribution of NBD-Labeled Phospholipids ((Cesar VOLTAGE-SENSITIVE DYE USING TWO-PHOTON EXCITATION Angeletti and J. Wylie Nichols.)) Department of Physiology. Emory University ((S.T. Hess, W.W. Webb.)) NIH Developmental Resource for Biophysical Imaging School of MIedicine. Atlanta GA 30322 and Opto-electronics, Cornell University, Ithaca, NY 14853 Dithionite quenching of the fluorescence of 'NBD-labeled lipids has been used to Fluorescent voltage-sensitive dyes have the potential for great utility in neuro- quantify their distribution and translocation across cellular and organellar mem- science if the sensitivity, i.e. the fractional change in fluorescence intensity (AF/F) branes. Assaying the extent of dithionite quenching provides a straightforward for a 100 mV depolarization, can be improved. We have measured the two-photon method for determining the fraction of NBD-lipid exposed to the outer leaflet of excitation cross-section for di-8-ANEPPS, a particularly bright, photostable styryl- membranes that are impermeant to dithionite. However. it appears that many. if class dye, using a pulsed Ti:Sapphire infared laser, and have chosen an appropriate not all, cellular membranes are relatively permeable to dithionite. The present work wavelength for excitation in cells. Using two-photon excitation, bright fluorescence describes a method in which the initial rate of dithionite quenching. rather than the was observed in HeLa cells. Preliminary results show a large AF/F (30-50%) upon extent of quenching, was used to determine the fraction of different NBD-labeled depolarization of the cells using 200 mM KCI. The increased response (compared phospholipids exposed to the outer leaflet. This method permits the estimation of with at best 10-15% per 100 mV using standard epifluorescence) may be explained by the translocation process even in experimental conditions where the membrane is the differences in quantum mechanical selection rules between one- and two-photon semi-permeable to dithionite. We studied the translocation of several NBD-labeled processes. (Sponsored by NIH grants RR07719 and RR04224 and NSF grant DIR phospholipids across two biological membranes: brush border membranes vesicles 88002787). (BBMV) from rabbit intestine and secretory vesicles (SV) from sec 6-4 mutant veast cells. In the case of BBMIV. several different NBD-phospholipids were translocated from the outer to inner leaflet in a matter of minutes and reached an equilibrium distribution of 50% inside and outside. This movement was inhibitable by N- bromosuccinimide. The translocation rate and distribution of head group labeled .NBD-lysophosphatidylethanolamine. having one titratable negative charge, could be modified in the presence of a pH gradient. The same NBD-phospholipids were also translocated across SV to roughly 50%, in both leaflets with the exception of NBD- phosphatidic acid which was passively distributed with 80%c in the inner leaflet. (supported by NIH grant GM352410) T'u-Pos323 Tu-Pos324 USE OF ACYL CHAIN SPECIFIC FRET PROBES TO DETECT EFFECT OF SOLVENT POLARITY AND LIPID CHEMICAL FLUID/FLUID PHASE SEPARATION IN BILAYERS RICH IN DHA STRUCTURE ON NAPHTHALENE DERIVATIVES EXCITATION AND CHOLESTEROL. ((A.C. Dumaual, L.J. Jenski, and W. Stillwell.)) SPECTRA. ((L. A. Bagatollil.2. T. Parasassil, G. D. Fidelio2. and E. Dept. of Physiology and Biophysics, IU School of Medicine, Indpls., IN 46202. Grattont.)) tLFD/Physics. UIUC. 1110 W. Green. Urbana. IL 61801-3080 and 2Dpto. Qca. Biol6gica-CIQUIBIC. U. de C6rdoba, C6rdoba. Argentina. Here we employ fluorescence resonance energy transfer (FRET) to test the hy- pothesis that acyl chain specific fluorescent probes may be used to detect fluid/fluid Professor Gregorio WVeber first synthesized several naphthalene derivatises for the phase separations in a model membrane composed of 18:0, 18:1 PC/18:0, 22:6 study of the fluorescence dipolar relaxation. Among them is the 2-dimethylamino- PC/cholesterol. Previous experiments have demonstrated that cholesterol interacts 6-lauroylnaphthalene (LAURDAN). schich became a popular membrane polarity poorly with docosahexaenoic acid (DHA, 22:6)-containing phospholipids compared probe. Beside the emission spectral shift due to dipolar relaxation occurring in to other unsaturated phospholipids such as 18:0, 18:1 PC. This implies that choles- polar solvents. this probe also shows variations of the excitation spectrum. in par- terol may induce lateral phase separation in DHA-rich membranes. Differential scan- ticular the appearance of a red excitation at about 390 nm band in polar solvents. ning calorimetry (DSC) experiments on our model bilayers indicate that fluid/fluid In ester phospholipids. LAURDAN excitation red band is particularly intense in separations can indeed occur resulting in DHA-rich, cholesterol-poor and DHA- the gel phase. while it displays a lower intensity in the liquid-crystalline phase of poor, cholesterol-rich phases. The FRET experiments were designed to confirm ester phospholipids. in both phases of ether phospholipids. glycosphyngolipids and the DSC findings. The fluorescent donor molecules used in the FRET experiments sphingomyelin. The occurrence and intensity of the red excitation band also de- were: NBD-16:0, 16:0 PE; NBD-18:1, 18:1 PE; NBD-18:0, 18:1 PE; and NBD-18:0, pends upon the residue in the position 2 of the naphthalene moiety. To explore this 22:6 PE. The acceptor molecules were rhodamine (Rh): Rh-16:0, 16:0 PE; Rh-18:1, last point. excitation spectra of the 2-hydroxy and 2-methoxv-6-laurovlnaphthalene 18:1 PE; Rh-18:0, 18:1 PE; and Rh-18:0, 22:6 PE. The tested hypothesis is that (LAURNt and LAtRMIEN. respectivelt) in solvents and in lipids were examined. if the FRET probes show significant acyl chain affinity, then probes will partition W\e also studied the features of the excitation spectrum of the 2-dimethylamino-6- into a lipid phase of similar acyl chain composition. Therefore, the gel state 16:0, propionvlnaphthalene (PRODAN) in bilayer lipids. where this probe senses a more 16:0 PE probes may partition into a liquid condensed, cholesterol-rich phase; the polar environment with respect to LAURDAN. Our results suggest the stabiliza- liquid crystalline state 18:1, 18:1 PE probes may partition into a liquid expanded, tion of the ground-state Lo conformation of the polar naphthalene moiety in the cholesterol-poor phase; the 18:0, 18:1 PE probes will partition into the 18:0, 18:1 polar environment. Supported by grants from the National Institutes of Health PC-rich phase; and the 18:0, 22:6 PE probes will partition into the DHA-rich phase. (RR03155) and and by CONICOR. SeCyT U-NC. Fundacion Antorchas and CON- These experiments demonstrate that the acyl chain specific FRET probes tested ICET. Argentina. show only partial acyl chain affinity, severely limiting their potential application. Tu-Pos325 Tu-Pos326 PARTITIONING OF FLUORESCENT LIPIDS BETWEEN MODEL FLAVONOLS - NEW FLUORESCENCE MEMBRANE PROBES FOR MEMBRANES ((G.W. Feigenson, J. Huang, J.T. Buboltz.)) Section of PHASE BEHAVIOR OF PHOSPHOLIPIDS. ((O.P.Bondar. Biochemistry, MIolecular and Cell Biology, Cornell University. Ithaca, NY 14853 V.G.Pivovarenko*, and E.S.Rowe.)) Department of Biochemistry and Molecular Biology, University of Kansas Medical School, VA Medical Center. Kansas City. The partition coefficient Kp was measured for a headgroup-labeled phospholipid, MO 64128 and *Kiev State University. Ukraine 252017 12-NBD-PE, and for NBD-labeled cholesterol, equilibrated between large unilamel- lar vesicles (LUV) of a series of phosphatidylcholines, some containing variable con- Two flavonols, 3-hydroxy-4'-dimethylaminoflavone (FME) and 3-hydroxy- 4' (15- centrations of cholesterol. Cholesterol could be incorporated into some liposomes azocrawn-5) flavone (FRC) have been investigated as new fluorescence probes to up to a mole fraction of about 0.67. Fluorescence quenching in one LUV population detect the phase behavior of dipalmitoylphosphatidylcholine and dipalmitoylphos- enabled finding the equilibrium concentration of the labeled lipid in the different phatidylethanol in the presence and absence of cholesterol. The fluorescence emis- LUV. Times required to reach near-equilibrium values ranged between a half day sion spectra of flavonols display high sensitivity to their microenvironment. The and several days. Reliable equilibrium concentrations were obtained by monitoring FCR and FME each exhibit a single fluorescence peak at 535 nm in water and 518 the approach to equilibrium starting from a concentration below and from a con- nm in ethanol, which corresponds to the emission of the normal state (N). The flu- centration above the ultimate values. Using (16:0,18:1)-PC as the reference lipid. orescence of both probes in hexane show a single band at 550 nm, due to emission Kp for 12-NBD-PC ranged from a high value of about 1.7 favoring (16:0.18:1)- of the phototautomer form (PT). In the solvents of medium polarity the probes PC over (16:1,16:1)-PC, to a low value of about 0.9, favoring (22:1,22:1)-PC over exhibit two peaks. The peak positions and the PT/N intensitv ratio have been used (16:0,18:1)-PC. The 12-NBD-PE partitioned strongly into LUV that contained a to estimate the degree of polarity and hydration of the probe environment in
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