gamma-cyclodextrins greatly enhance translocation of hydrophobic fluorescent phospholipids from vesicles to cells in culture. Importance of molecular hydrophobicity in phospholipid trafficking studies.
AuthorsTanhuanpää K, Somerharju P
JournalJ Biol Chem
PubMed ID10585403
'Short-chain, fluorescent derivatives are commonly used to investigate intracellular phospholipid trafficking. However, their use can yield misleading results because they, unlike the native species, can rapidly distribute between organelles due to their low hydrophobicity. On the other hand, hydrophobic derivatives are very difficult to introduce to cells and thus have ... More
Changes in the lipid dynamics of liposomal membranes induced by poly(ethylene glycol): free volume alterations revealed by inter- and intramolecular excimer-forming phospholipid analogs.
AuthorsLehtonen JY, Kinnunen PK
JournalBiophys J
PubMed ID8075332
'Influence of osmotic shrinkage, swelling, and dehydration on large unilamellar liposomes (LUVs) of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) was investigated using the fluorescent lipid probes 1-palmitoyl-2-[10-(pyren-1-yl)]-decanoyl-sn-glycero-3-phosphocholi ne (PPDPC) and 1,2-bis[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC). Increasing concentrations of poly(ethylene glycol) (PEG, average molecular weight of 6000) producing osmotic gradients delta omega up to 250 mOsm/kg were first ... More
Binding and dissociation of cytochrome c to and from membranes containing acidic phospholipids.
AuthorsSubramanian M, Jutila A, Kinnunen PK
JournalBiochemistry
PubMed ID9477968
'Membrane association and detachment of cytochrome c (cyt c) in millisecond to second time domain were investigated by stopped-flow fluorescence spectroscopy monitoring the efficiency of energy transfer from a pyrene-fatty acid containing phospholipid derivative, 1-palmitoyl-2-[10-(pyren-1-yl)-decanoyl]-sn-glycero-3-phosphoglyce rol (PPDPG, mole fraction X = 0.01) to the heme of the cyt c. Large ... More
Continuous, vesicle-based fluorimetric assays of 14- and 85-kDa phospholipases A2.
AuthorsBayburt T, Yu BZ, Street I, Ghomashchi F, Laliberté F, Perrier H, Wang Z, Homan R, Jain MK, Gelb MH
JournalAnal Biochem
PubMed ID8600835
'This paper describes the synthesis and analysis of new substrates for the 85-kDa, mammalian, cytosolic phospholipase A2 (cPLA2) and the 14-kDa, human nonpancreatic, secreted phospholipase A2 (sPLA2). Phosphatidylcholines containing an arachidonyl chain at the sn-2 position and either a 10-pyrenedecyl or a 10-pyrenedecanoyl chain at the sn-1 position were synthesized ... More
Real-time fluorescence polarization measurements: interaction of phospholipase A2 with a fluorescent lecithin derivative.
AuthorsMonti JA, McBride MR, Barker SA, Linton JL, Christian ST
JournalJ Biochem Biophys Methods
PubMed ID3924989
'We have modified an SLM 4800 fluorometer to allow continuous measurement of fluorescence polarization. The modifications included construction of simple mechanical and electronic accessories which allowed polarizer position to be program-controlled by an HP 9815 calculator. With these modifications we were able to follow the interaction of the fluorescent lipid ... More
Binding of DNA to liposomes containing different derivatives of sphingosine.
AuthorsKõiv A, Kinnunen PK
JournalChem Phys Lipids
PubMed ID7923481
'Binding of DNA to dimyristoylphosphatidylcholine (DMPC) liposomes containing different sphingosine derivatives was investigated. DNA labelled with adriamycin was used as a fluorescence quencher and its membrane association was observed by resonance energy transfer from liposomes incorporating a pyrene-derivatized lipid bisPDPC as a donor and containing 19 mol% of sphingosine, dihydro-, ... More
Sphingosine-mediated membrane association of DNA and its reversal by phosphatidic acid.
AuthorsKinnunen PK, Rytömaa M, Kõiv A, Lehtonen J, Mustonen P, Aro A
JournalChem Phys Lipids
PubMed ID7509731
'Resonance energy transfer was measured between egg phosphatidylcholine liposomes containing the intramolecular excimer forming pyrene-labelled phospholipid analogue 1,2-bis[pyren-1-(-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC) as a donor and DNA-bound adriamycin as an acceptor. Membrane association of DNA turned out to be critically dependent on the presence of sphingosine in the liposomes. Identical result was obtained ... More
Detection and characterization of the onset of bilayer packing defects by nanosecond-resolved intramolecular excimer fluorescence spectroscopy.
AuthorsCheng KH, Somerharju P, Sugar I
JournalChem Phys Lipids
PubMed ID7820901
'Bilayer packing defects in binary dilinoleoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylcholine (DLPE/POPC) lipid mixtures have been studied by the use of nanosecond-resolved intramolecular excimer fluorescence spectroscopy. Frequency-domain fluorescence intensity decays of dual-chain labelled dipyrenyl lipids of different chain lengths in DLPE/POPC mixtures were acquired at both the monomer (392 nm) and excimer (475 ... More
Intramolecular excimer kinetics of fluorescent dipyrenyl lipids: 1. DMPC/cholesterol membranes.
AuthorsCheng KH, Ruymgaart L, Liu LI, Somerharju P, Sugar IP
JournalBiophys J
PubMed ID7948704
'The intramolecular dynamics of the excimer forming dipyrenyl lipids (DipynPC) of different chain lengths (n) in ethanol and in dimyristoylphosphatidycholine (DMPC) membranes was investigated by the use of frequency-domain fluorescence intensity decay technique. Based on a 3-state model, the extent of aggregation and rotational rate of the two intralipid pyrene ... More
Activity of phospholipase A2 on a fluorescent substrate incorporated into non-hydrolyzable phospholipid liposomes.
AuthorsFernández MS, Juárez JA
JournalBiochim Biophys Acta
PubMed ID8204643
'The activity of phospholipase A2 (PLA2) on phospholipid liposomes depends on the physicochemical properties of the aggregated substrate, which are subject to continuous modification by the products released during hydrolysis. We propose here an experimental design that, by means of the incorporation of a fluorescent substrate at very low molar ... More
Oxidation of unsaturated phospholipids in membrane bilayer mixtures is accompanied by membrane fluidity changes.
AuthorsBorst JW, Visser NV, Kouptsova O, Visser AJ
JournalBiochim Biophys Acta
PubMed ID10962288
'Steady-state and time-resolved fluorescence spectroscopy has been used to obtain information on oxidation processes and associated dynamical and structural changes in model membrane bilayers made from single unilamellar vesicles (SUV''s) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) mixed with increasing amounts of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC). The highly unsaturated arachidonoyl chain containing four double bonds is ... More
The interaction between protein kinase C and lipid cofactors studied by simultaneous observation of lipid and protein fluorescence.
AuthorsPap EH, van den Berg PA, Borst JW, Visser AJ
JournalJ Biol Chem
PubMed ID7836388
'The interaction of protein kinase C (PKC) with lipids was probed by a dual approach. Pyrene-labeled lipid analogues of diacylglycerol, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylcholine (PC) were used both as acceptors of tryptophan excitation energy of PKC and as membrane probes for intra- and intermolecular lipid ... More
Pyrene-labeled lipids as tools in membrane biophysics and cell biology.
AuthorsSomerharju P
JournalChem Phys Lipids
PubMed ID12093535
Pyrene is one of the most frequently used lipid-linked fluorophores. Its most characteristic features are a long excited state lifetime and (local) concentration-dependent formation of excimers. Pyrene is also hydrophobic and thus does not significantly distort the conformation of the labeled lipid molecule. These characteristics make pyrene lipids well-suited for ... More
Intramolecular excimer kinetics of fluorescent dipyrenyl lipids: 2. DOPE/DOPC membranes.
AuthorsCheng KH, Ruymgaart L, Liu LI, Somerharju P, Sugar IP
JournalBiophys J
PubMed ID7948705
The intramolecular dynamics of the excimer-forming dipyrenyl lipids (DipynPE) of different chain lengths (n) in fully hydrated dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) binary mixtures was investigated by the use of frequency-domain fluorescence intensity dcay technique. Using a 3-state model (see companion paper), the extent of aggregation and rotational rate of ... More
Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.
AuthorsXu C, Zipfel W, Shear JB, Williams RM, Webb WW
JournalProc Natl Acad Sci U S A
PubMed ID8855254
Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's ... More
Fluorescence studies of lipid regular distribution in membranes.
AuthorsChong PL, Sugár IP
JournalChem Phys Lipids
PubMed ID12093540
This article reviews the use of fluorescent lipids and free probes in the studies of lipid regular distribution in model membranes. The first part of this article summarizes the evidence and physical properties for lipid regular distribution in pyrene-labeled phosphatidylcholine (PC)/unlabeled PC binary mixtures as revealed by the fluorescence of ... More
Evidence for ternary complex formation by histone H1, DNA, and liposomes.
AuthorsKõiv A, Palvimo J, Kinnunen PK
JournalBiochemistry
PubMed ID7794914
Using three different donor-acceptor pairs for resonance energy transfer, interactions in systems composed of histone H1, liposomes, and DNA were investigated. While weak attachment of H1 to phosphatidylcholine (PC) liposomes was observed, the inclusion of phosphatidylserine (PS), phosphatidylglycerol (PG), or phosphatidic acid (PA) strongly enhanced the membrane association of H1, ... More
Evidence for the extended phospholipid conformation in membrane fusion and hemifusion.
AuthorsHolopainen JM, Lehtonen JY, Kinnunen PK
JournalBiophys J
PubMed ID10096906
Molecular-level mechanisms of fusion and hemifusion of large unilamellar dioleoyl phosphatidic acid/phosphocholine (DOPA/DOPC, 1:1 molar ratio) vesicles induced by millimolar Ca2+ and Mg2+, respectively, were investigated using fluorescence spectroscopy. In keeping with reduction of membrane free volume Vf, both divalent cations increased the emission polarization for 1,6-diphenyl-1,3, 5-hexatriene (DPH). An ... More
Fluorescence-based assays of lipases, phospholipases, and other lipolytic enzymes.
AuthorsHendrickson HS
JournalAnal Biochem
PubMed ID8059934
In choosing an assay, one needs to consider the following questions: What level of sensitivity is required? Must the assay be continuous? Is the substrate readily available; can it be purchased or must it be custom synthesized? How specific is the substrate? How convenient is the method? How compatible is ... More
Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer.
AuthorsCurran AR, Templer RH, Booth PJ
JournalBiochemistry
PubMed ID10413507
Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon ... More
Role of lateral phase separation in the modulation of phospholipase A2 activity.
AuthorsBurack WR, Yuan Q, Biltonen RL
JournalBiochemistry
PubMed ID8422369
Phospholipase A2-catalyzed hydrolysis of phosphatidylcholine large unilamellar vesicles is characterized by a period of slow hydrolysis followed by a rapid increase in the rate of hydrolysis. The temporal relationship between the burst of PLA2 activity and the lateral distribution of substrate and product lipids was examined by simultaneously recording product ... More
Direct, ultrasensitive, and selective optical detection of protein toxins using multivalent interactions.
AuthorsSong X, Swanson BI
JournalAnal Chem
PubMed ID10366891
Three highly sensitive, selective, and reagent-free optical signal transduction methods for detection of polyvalent proteins have been developed by directly coupling distance-dependent fluorescence self-quenching and/or resonant-energy transfer to the protein-receptor binding events. The ganglioside GM1, as the recognition unit for cholera toxin (CT), was covalently labeled with fluorophores and then ... More
Quantitative analysis of lipid-lipid and lipid-protein interactions in membranes by use of pyrene-labeled phosphoinositides.
AuthorsPap EH, Hanicak A, van Hoek A, Wirtz KW, Visser AJ
JournalBiochemistry
PubMed ID7619810
The lateral and rotational dynamics of phosphoinositides and their interactions with proteins were characterized using pyrene-labeled lipid analogues. In these systems, the collision frequency of pyrene-labeled lipids was studied by monitoring the monomeric pyrene fluorescence yield as a function of their mole fraction in the membranes. From this dependence, the ... More
Correlation between bilayer lipid dynamics and activity of the diglucosyldiacylglycerol synthase from Acholeplasma laidlawii membranes.
AuthorsKarlsson OP, Rytömaa M, Dahlqvist A, Kinnunen PK, Wieslander A
JournalBiochemistry
PubMed ID8756472
In the single membrane of Acholeplasma laidlawii a specific glucosyltransferase synthesize the major, lamellar-forming lipid diglucosyldiacylglycerol (DGlcDAG) from the major, nonlamellar-prone monoglucosyldiacylglycerol (MGlcDAG). This is crucial for the maintenance of phase equilibria close to a bilayer-nonbilayer transition and a nearly constant spontaneous curvature in the membrane lipid bilayer. Acyl chain ... More
Lateral organization of liquid-crystalline cholesterol-dimyristoylphosphatidylcholine bilayers. Evidence for domains with hexagonal and centered rectangular cholesterol superlattices.
AuthorsVirtanen JA, Ruonala M, Vauhkonen M, Somerharju P
JournalBiochemistry
PubMed ID7547888
The lateral organization of fluid cholesterol-dimyristoylphosphatidylcholine (DMPC) bilayers was studied by measuring the response of fluorescent membrane probes, dipyrenylphosphatidylcholines (diPyrxPCs) or merocyanine 540, to the variation of cholesterol concentration. Parallel absorbance and light-scattering measurements were also carried out. The excimer-to-monomer ratio of diPyrxPCs displayed abrupt deviations at particular cholesterol mole ... More
Dipyrenylphosphatidylcholines as membrane fluidity probes. Pressure and temperature dependence of the intramolecular excimer formation rate.
AuthorsSassaroli M, Vauhkonen M, Somerharju P, Scarlata S
JournalBiophys J
PubMed ID8431538
We have measured the pressure dependence of the intramolecular excimer formation rate, K(p), for di-(1'-pyrenedecanoyl)-phosphatidylcholine (dipy10PC) probes in single-component lipid multilamellar vesicles (MLV) as a function of temperature. Apparent volumes of activation (V(a)) for intramolecular excimer formation are obtained from the slopes of plots of log K(p) versus P. For ... More