Steady-state Fluorescence Polarization Measurements Research Articles

Fluorescence life-time imaging and steady state polarization for examining binding of fluorophores to gold nanoparticles.

Nanocomposites as multifunctional agents are capable of combing imaging and cell biology technologies. The conventional methods used for validation of the conjugation process of nanoparticles (NPs) to fluorescent molecules such as spectroscopy analysis and surface potential measurements, are not sufficient. In this paper we present a new and highly sensitive procedure that uses the combination of (1) fluorescence spectrum, (2) fluorescence lifetime, and (3) steady state fluorescence polarization measurements. We characterize and analyze gold NPs with Lucifer yellow (LY) surface coating as a model. We demonstrate the ability to differentiate between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes. We suggest the approach for neuroscience applications where LY is used for detecting and labeling cells, studying morphology and intracellular communications. Histograms of Fluorescence lifetime imaging (FLIM) of free LY dye (Left) in comparison to the conjugated dye to gold nanoparticles, LY-GNP (Middle) enable the differentiation between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes (Right).

Steady-state polarization from cylindrically symmetric fluorophores undergoing rapid restricted motion

Steady-state fluorescence polarization measurements provide a relatively simple method for investigating the orientation of molecular components in ordered biological systems. However, the observed fluorescence polarization ratios also depend on any mobility of the fluorophores on the time scale of the fluorescence lifetime. Such mobility commonly arises from incomplete immobilization of a fluorescent probe on the macromolecule of interest. A theoretical formalism is presented for the interpretation of steady-state fluorescence polarization ratios in the presence of such rapid fluorophore motion. It is assumed that the fluorophores move freely within a cone between absorption and emission of a photon. Only one new parameter is introduced to describe fluorophore motion, the semi-angle of the cone, and this can be measured in separate experiments on an isotropic system. The fluorophore orientations are assumed to have cylindrical symmetry, but the symmetry axis need not be in the same plane as the center axes of the absorption and emission cones. This geometry applies to muscle and other biological fibers.

Photophysical Properties of Ruthenium Polypyridyl Photonic SiO2 Gels

The photophysical properties of Ru(bpy)[sub 3](PF[sub 6])[sub 2] and cis-Ru(bpy)[sub 2](CN)[sub 2] (where bpy = 2,2[prime]-bipyridine) doped into monolithic SiO[sub 2] aquo and alco gels prepared by sol-gel processing techniques is described. The excited-state properties of the dopants are dramatically altered by the gel matrix. The photoluminescence emission energy and quantum yield of the dopants increase through the sol-to-gel transition. Steady-state fluorescence polarization measurements reveal significant anisotropy. Time-resolved fluorescence decays observed from these photonic materials are complex. Evidence for a bimodal distribution of dopant sites is obtained from modeling the emission decays and from application of the exponential series method. Implications of these results for molecular photonic applications are discussed. 31 refs., 7 figs., 1 tab.

Erythrocyte membrane lateral sterol domains: A dehydroergosterol fluorescence polarization study

Structural domains of cholesterol and their regulation in the erythrocyte membrane are poorly understood. Dehydroergosterol fluorescence polarization change was used to continuously monitor the kinetics of sterol exchange and sterol domain size in erythrocyte ghost membranes. Direct correlation between molecular sterol exchange and steady-state dehydroergosterol fluorescence polarization measurements was obtained without separation of donor and acceptor membranes. Three important observations were made. First, sterol exchange between small unilamellar vesicles (SUV) with the same cholesterol/phospholipid ratio as the erythrocyte membrane (1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol = 1:1) was resolved into three kinetic cholesterol domains: 23 +/- 9% of total sterol was rapidly exchangeable, with t1/2 = 23 +/- 6 min; 59 +/- 9% of total sterol was slowly exchangeable, with t1/2 = 135 +/- 3 min; and 19 +/- 9% of total sterol was essentially nonexchangeable, with a t1/2 of days. Second, the substitution of erythrocyte ghosts for SUV as an acceptor significantly altered the kinetic parameters of sterol exchange from donor SUV, graphically showing that both the properties of the acceptor and spontaneous desorption of cholesterol from the donor SUV influenced spontaneous cholesterol transfer. Third, studies of exchange between erythrocyte ghosts revealed multiple kinetic pools of sterol differing from those in the SUV: 4 +/- 2% of total sterol was rapidly exchangeable, with t1/2 = 32 +/- 9 min; 29 +/- 3% of total sterol was very slowly exchangeable, with t1/2 = 23 +/- 7 h; and a surprisingly large 67 +/- 2% of total sterol was nonexchangeable, with a t1/2 of days.

Sterol domains in phospholipid membranes: dehydroergosterol polarization measures molecular sterol transfer

The domain structure of cholesterol in membranes and factors affecting it are not well understood. A method, based on kinetics of Δ5,7,9,(11),22-ergostatetraen-3β-ol (dehydroergosterol) fluorescence polarization change and not requiring separation of donor and acceptor membranes, was used to examine sterol domains in three-component cholesterol: dehydroergosterol: phospholipid small unilamellar vesicles (SUV). A new mathematical data treatment was developed to provide a direct correlation between molecular sterol exchnage and steady-state dehydroergosterol fluorescence polarization measurements. The method identified multiple kinetic pools of sterol in SUV: a small but rapidly exchanging pool, a predominant slowly exchanging pool, and a very slowly exchangeable (nonexchangeable) pool. The relative sizes of the pools and half-times of exchange were highly dependent on the presence of acidic phospholipids and on cytosolic proteins involved in sterol transfer. Thus, the method provides a direct measure of molecular sterol transfer between membranes without separating donor and acceptor membranes.

Thermotropic Properties of Cellular Membranes in Dormant and Non-DormantEchinochloa crus-galli(L.) Beauv. Seeds

Steady-state fluorescence polarization measurements with 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to monitor thermotropic transitions in microsomal fractions and plasma membrane vesicles isolated from barnyardgrass seeds during the transition from dormancy to germination

Relation between membrane phospholipid composition, fluidity and function in mitochondria of rat brown adipose tissue. Effect of thermal adaptation and essential fatty acid deficiency

Male weanling rats were maintained either at 28° C (thermoneutrality) or at 5° C (cold adaptation). During 9 weeks they were fed either a 2% hydrogenated coconut oil diet deficient in essential fatty acids or a diet containing 2% sunflower oil. The respective incidences of cold adaptation and of EFA deficiency on lipid composition of mitochondrial membranes from brown adipose tissue (BAT) were investigated. Using 1,6 diphenylhexatriene (DPH) as a probe, the parameters of membrane fluidity were estimated by steady-state fluorescence polarization measurements ( r s ) and by time-resolved fluorescence anisotropy decay (order parameter S). Cold acclimation induced a decrease of phosphatidylcholine to phosphatidylethanolamine (PC/PE ratio), an increase of the total fatty acid unsaturation index (T.U.). EFA deficiency had the same effect as cold on the PC/PE ratio, but decreased T.U. Cold adaptation induced a larger decrease of S than of r s , whereas EFA deficiency only increased r s and did not modify S. In liposomes prepared from mitochondrial lipids, r s values were smaller than in whole mitochondria. Both in cold-adapted and in EFA-deficient rats the variations of r s were correlated with lipid unsaturation. Comparison between BAT thermogenic activity, assessed by GDP binding and proportions of PE and PC showed a high correlation suggesting a change in the membrane occurring with the increase of mitochondrial activity that could be related to phospholipid composition rather than to membrane fluidity.

Influence of N, N-dimethylaniline on the association of phenobarbital-induced cytochrome P-450 and NADPH-cytochrome c( P-450) reductase in a reconstituted rabbit liver microsomal enzyme system

N, N-Dimethylaniline when added to reaction mixtures provokes deviation from Michaelis-Menten law of the interaction kinetics of NADPH-cytochrome c( P-450) reductase (NADPH: ferrihaemoprotein oxidoreductase, EC 1.6.2.4) with highly purified phenobarbital-induced rabbit liver microsomal cytochrome P-450 ( P-450LM 2). This phenomenon is not associated with the low-to-high spin transition in the iron-coordination sphere of the haemoprotein, as elicited by the arylamine. Substrate-triggered departure from linearity of the kinetics is abolished by inclusion into the assay media of p-chloromercuribenzoate hinting at a vital role in the process of thiols. Similarly, the parabolic progress curve ( n H = 1.7) is transformed to a straight line ( n H = 1.01) when the N-terminal reductase-binding domain in the P-450LM 2 molecule is selectively blocked through covalent attachment of fluorescein isothiocyanate (FITC); such a modification does not alter the affinity of the haemoprotein for the amine substrate. Steady-state fluorescence polarization measurements reveal that N, N-dimethylaniline perturbs the motional properties of the fluorophore-bearing reductase-binding region, suggesting the induction of a conformational change. Summarizing these results, the data possibly indicate N, N-dimethylaniline-induced cooperativity in the association of reductase with P-450LM 2.

Quantitative contributions of cholesterol and the individual classes of phospholipids and their degree of fatty acyl (un)saturation to membrane fluidity measured by fluorescence polarization.

Steady-state fluorescence polarization (P) measurements, using the probe 1,6-diphenyl-1,3,5-hexatriene, in a large variety of well-defined liposomes at 25 degrees C allowed a quantitative description of the contributions of cholesterol, sphingomyelin, and (un)saturation of fatty acyl groups in the various phospholipids to the structural order (or the mutual affinity) of membrane lipids. The P values for liposomes prepared from lipid extracts of natural (purified) membranes of various origins could be more or less predicted (calculated) from the relative contributions of the individual lipid components. In all cases, the polarization varied with the cholesterol/phospholipid molar ratio (C/PL) according to the equation P = Pplat -(Pplat -Pzero) exp(-alpha C/PL), in which Pzero refers to the polarization without cholesterol and Pplat is a maximal plateau value, reached at a high C/PL (greater than 1). The "cholesterol-ordering coefficient" alpha of the phospholipids was found to increase with the fraction of sphingomyelin or dipalmitoylphosphatidylcholine molecules, indicating that the susceptibility of phospholipids to be ordered by cholesterol is increased by these compounds. Pzero increases curvilinearly with the fraction of either of these molecules. Pplat increases linearly with the fraction of saturated acyl chains for most phospholipids. Highly unsaturated fatty acyl chains (e.g., 20:4 and 22:6) strongly depress Pplat but not Pzero. The results suggest that such phospholipids are unlikely to associate with cholesterol and may thus create extremely fluid membrane domains. The disproportionation of cholesterol in the cell can be understood by the differing composition of the phospholipids in plasma membranes and endomembranes and their ordering susceptibility (affinity) toward cholesterol.

A cell membrane correlate of tardive dyskinesia in patients treated with phenothiazines.

Phenothiazine administration to psychiatric patients is associated with an increase in the "structural order" of platelet membranes as determined by steady-state fluorescence polarization measurements with 1,6-diphenyl-1,3,5-hexatriene (DPH), a fluorescent probe that localizes preferentially in the hydrocarbon region of cell membranes (Zubenko and Cohen 1984, 1985a, b). In this study, platelet membranes prepared from a group of psychiatric patients who developed tardive dyskinesia following chronic treatment with phenothiazines exhibited a significant elevation in DPH fluorescence polarization when compared to similar preparations from an otherwise matched group of patients who had no symptoms or history of tardive dyskinesia. The distribution of polarization values obtained for the tardive dyskinesia group displayed minimal overlap with that of an unmedicated, psychiatrically-healthy control group matched for age and gender. The fluorescence polarization of DPH-labelled platelet membranes was not significantly correlated with phenothiazine daily dose or serum cholesterol concentration in the phenothiazine-treated patient groups, or with dyskinesia severity (AIMS rating) in the tardive dyskinesia group. Patient gender and the presence of an affective disorder did not significantly correlate with DPH fluorescence polarization. The potential physiological and clinical significance of these findings is discussed.

Deuterium isotope effect on the stability of molecules: phospholipids

To examine the deuterium isotope effect on the stability of phospholipids, the phase transition properties of bilayer vesicles, prepared from deuterated phospholipids, were investigated and compared with those of nondeuterated (normal) phospholipids. Three lipids, 1,2-distearoyl-, 1,2-dipalmitoyl-, and 1,2-dimyristoylphosphatidylcholines, were studied. Differential scanning microcalorimetry showed that, unlike a sharp phase transition observed in a nondeuterated lipid, a deuterated lipid exhibits a broad transition which is associated with a lower temperature (t/sub m/) and small enthalpy (..delta..H/sub m/) and entropy (..delta..S/sub m/) of the transition. The magnitude of t/sub m/ in deuterated lipid is 3-5/sup 0/C lower than in the corresponding nondeuterated lipid. Deuteration causes the magnitude of ..delta..H/sub m/ and ..delta..S/sub m/ to be decreased by 14-35%. For each CH/sub 2/ group, the deuterium substitution in general decreases ..delta..H/sub m/, ..delta..G/sub m/ (Gibbs energy), and ..delta..S/sub m/ by 272 J/mol, 15.4 J/mol, and 0.7 J/mol deg), respectively. Steady-state fluorescence polarization (P) measurements have demonstrated that, at a temperature above t/sub m/ (liquid-crystalline phase), deuterated and nondeuterated lipids essentially have a same magnitude of P, while at a temperature below t/sub m/ (gel phase), deuterated lipid has a lower P than nondeuterated lipid. Consequently, deuterated lipid has a lower magnitude of ..delta..P more » (difference in P below t/sub m/), indicating that the structural differences between gel and liquid-crystalline phase is less profound in deuterated lipids. The melting properties of deuterated and nondeuterated fatty acids corresponding to these three lipids were also examined. 20 references, 3 figures, 1 table. « less

Effects of phenothiazine treatment on the physical properties of platelet membranes from psychiatric patients

Exposure of normal platelet membranes in vitro to clinically relevant concentrations of phenothiazines has been reported to result in an increase in the structural order of the hydrocarbon region of these membranes (Zubenko and Cohen 1984b,c). In this study, the order parameters (reciprocal of fluidity) of platelet membranes prepared from psychiatric patients receiving phenothiazine antipsychotic drugs were compared to those of an otherwise matched population of psychiatric patients who were not receiving medications that significantly affect this cell membrane property in vitro (Zubenko and Cohen 1984b,c). Membrane order was determined ex vivo by steady-state fluorescence polarization measurements employing 1,6-diphenyl 1,3,5-hexatriene, a fluorescent probe that localizes preferentially in the hydrocarbon region of cell membranes. Phenothiazine administration was associated with a significant increase in platelet membrane order and most likely results from a direct action of these agents on cell membranes. The platelet membrane order parameters of phenothiazine-free patients did not differ significantly from those of a matched group of unmedicated, normal controls. Platelet membrane order was not correlated with daily phenothiazine dose or serum cholesterol concentration. Patient gender did not significantly affect platelet membrane order in any of the groups studied. The potential physiological and clinical significance of phenothiazine-induced increases in cell membrane order is discussed.

The effects of pressure and cholesterol on rotational motions of perylene in lipid bilayers

Using steady-state fluorescence polarization measurements, an isothermal pressure-induced phase transition was observed in dimyristoyl- l-α-phosphatidylcholine multilamellar vesicles containing perylene. The temperature-to-pressure equivalence, d T/d P, estimated from the phase transition pressure, P 1 2 , is about 22 K · kbar −1, which is comparable to values determined from diphenylhexatriene polarization (Chong, P.L.-G. and Weber, G. (1983) Biochemistry 22, 5544–5550). In addition, we have employed a new method, introduced in this paper, to calculate the rate of in-plane rotation ( R ip) and the rate of out-of-plane rotation ( R op) of perylene in lipid bilayers. The effects of pressure and cholesterol on the rotational rates of perylene in two lipid bilayer systems have been examined. They are 1-palmitoyl-2-oleoyl- l-α-phosphatidylcholine (POPC) multilamellar vesicles (MLV) and 50 mol% cholesterol in POPC (MLV). R op is smaller than R op due to the fact that the out-of-plane rotation requires a larger volume change than the in-plane rotation. Cholesterol seems not to affect R op significantly, but pressure causes a decrease in R op by about a factor of three. In contrast, the effects of pressure and cholesterol on R ip are less straightforward. At 1 atm cholesterol increases R ip by a factor of about two. Similarly, in the absence of cholesterol, 1.5 kbar pressure essentially triples R ip. However, if both cholesterol is added and pressure is applied, R ip decreases sharply. The possible interactions between cholesterol and perylene are discussed.

Effects of psychotropic agents on the physical properties of platelet membranes in vitro.

Normal platelet membranes were exposed in vitro to a variety of psychotropic medications commonly used in the treatment of patients with psychiatric disorders. Changes in structural order at the hydrocarbon region of the drug-exposed membranes were determined by steady-state fluorescence polarization measurements employing the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Chlorpromazine, an aliphatic phenothiazine, produced a significant increase in DPH fluorescence polarization at concentrations from 2-200 microM. Thioridazine, a piperidine phenothiazine, and three piperazine derivatives, perphenazine, trifluoperazine, and fluphenazine, produced significant increases in this parameter at concentrations from 20-200 microM. The other agents tested, including thiothixene, lithium, antidepressants, anxiolytics, and anticonvulsants, were without effect in the concentration ranges examined. The phenothiazine-induced increase in DPH fluorescence polarization apparently depended on the structure of the phenothiazine nucleus; changes in side-chain structure appeared to modulate this effect, most likely by altering the inherent membrane solubility of the agents.

In vitro effects of psychotropic agents on the microviscosity of platelet membranes.

Normal platelet membranes were exposed in vitro to a variety of psychotropic agents, including chlorpromazine, fluphenazine, haloperidol, imipramine, and lithium. Changes in microviscosity of the hydrocarbon layer of the drug-exposed membranes were determined by steady-state fluorescence polarization measurements, employing the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH). Concentrations of the phenothiazines chlorpromazine and fluphenazine above 200 nM produced significant increases in microviscosity, while haloperidol produced a small but consistent decrease in microviscosity in the concentration range of 200 nM to 200 microM. Imipramine and lithium were without effect. The phenothiazine-induced increase in microviscosity was apparently dependent upon the structure of the phenothiazine nucleus; side chain structure was less important to this effect.

Site-specific fluorescein-labeled cobra alpha-toxin. Biochemical and spectroscopic characterization.

Cobra alpha-toxin purified from Naja naja siamensis venom was labeled with near stoichiometric quantities of fluorescein isothiocyanate. A monofluorescein alpha-toxin was separated in 50-60% yield from unconjugated alpha-toxin and other reaction products by ion exchange chromatography. The isolated mono-conjugated alpha-toxin electrofocuses largely as a single entity with 92% appearing with a pI of 9.6. The unmodified toxin has a pI of 10.7. Thermolysin digestion and subsequent high pressure liquid chromatography of the peptides yield two dominant fluorescent peaks, both of which can be traced to the labeling of lysine 23. The NE-fluorescein isothiocyanate (FITC)-Lys-23 alpha-toxin shows an apparent reduction in quantum yield when compared with either free FITC or the denatured and reduced NE-FITC-Lys-23 alpha-toxin. The reduction of fluorescence is likely to be due to static quenching of the fluorescein by the tryptophanyl and tyrosyl residues (25 and 21, respectively) in the "central loop" region. Binding of the NE-FITC-Lys-23 alpha-toxin to the membrane-associated acetylcholine receptor is accompanied by a 95 +/- 22% increase in fluorescence which probably reflects perturbation of the beta-pleated sheet character of the region containing residues 20-25 of the alpha-toxin. Steady state fluorescence polarization measurements of NE-FITC-Lys-23 alpha-toxin yield a rotational correlation time of 3.7 ns, suggesting FITC is largely immobilized on the alpha-toxin. The NE-FITC-Lys-23 alpha-toxin binds with a dissociation constant of 4 nM determined by fluorescence polarization.

Steady-state fluorescence polarization of dansylcadaverine-fibrinogen: evidence for flexibility

The conformation of fibrinogen in solution has been investigated by steady-state fluorescence polarization measurements. Factor XIIIa has been employed to enzymatically incorporate 1-6 mol of dansylcadaverine/mol of fibrinogen into a specific glutamine residue near the carboxy terminus of the gamma chain and up to two sites on the alpha chain. The fluorescence emission maximum of the labeled protein is shifted to 495 nm (from 538 nm for the fluorophore in solution) and the intensity substantially enhanced, indicating the covalently linked dansyl groups residue in a hydrophobic environment in the interior of the protein. This covalent modification does not interfere with the formation of fibrin, following thrombin activation. Steady-state fluorescence polarization measurements were carried out as a function of temperature and in high viscosity solvents. The fluorescent lifetime of dansylcadaverine-fibrinogen was determined by a phase shift technique. Analysis of the data by the Perrin-Weber treatment yields a rotational relaxation time of 160 ns, considerably faster than any realistic hydrodynamic model of fibrinogen would predict. The results are discussed in terms of segmental flexibility.

Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements

This paper presents an interpretation of fluorescence polarization measurements in lipid membranes which are labelled with the apolar probe 1,6-diphenyl-1,3,5-hexatriene. The steady-state fluorescence anisotropy, r S , is resolved into a fast decaying or kinetic component, r f , and an infinitely slow decaying or static component, r ∞ . The latter contribution, which predominates in biological membranes, is exclusively determined by the degree of molecular packing (order) in the apolar regions of the membrane; r ∞ is proportional to the square of the lipid order parameter. An empirical relation between r S and r ∞ is presented, which is in agreement with a prediction based on a theory of rotational dynamics in liquid crystals. This relation enabled us to estimate a lipid structural order parameter directly from simple steady-state fluorescence polarization measurements in a variety of isolated biological membranes. It is shown that major factors determining the order parameter in biomembranes are the temperature, the cholesterol and sphingomyelin content and (in a few systems) the membrane intrinsic proteins.

Rapid membrane response during low-temperature acclimation Correlation of early changes in the physical properties and lipid composition of Tetrahymena microsomal membranes

When Tetrahymena pyriformis cells grown at 39°C were chilled to 15°C, a rapid desaturation of microsomal phospholipid-bound fatty acids was observed. A concurrent but even more rapid change in the physical properties of the microsomal lipids was detected by steady-state fluorescence polarization measurements of the probe 1,6-diphenyl-1,3,5-hexatriene in lipid multilamellar vesicles. Whereas polarization vs. temperature plots of lipids from 39°C-grown cells showed discrete break points (abrupt slope changes thought to indicate altered phase separation rates) at characteristic temperatures, plots made using lipids from equivalent cells chilled to 15°C for 15 or 20 min lacked such clearly defined break points. The sharp break points reappered in plots of microsomal lipids from cells maintained at 15°C for 30 min or longer, but in these curves the temperature of each break point was several degrees lower than in 39°C-cell lipids and nearly the same as in cells fully acclimated to low temperature (48 h or more at 15°C). Fluorescence polarization studies on mixtures of natural lipids from 39°C-cells and cells shifted to 15°C or on mixtures of natural and synthetic lipids revealed that each of the two break points in a polarization vs. temperature plot can respond to changes in lipid composition independently of the other. It is concluded that the expeditious desaturation of certain key fatty acids, perhaps coupled with limited retailoring of phospholipid molecular species, leads to pronounced physical changes in Tetrahymena microsomal membranes as the first step of low temperature acclimation.

Discontinuous thermotropic response of Tetrahymena membrane lipids correlated with specific lipid compositional changes

Steady-state fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene in microsomal lipids from Tetrahymena pyriformis cells grown at 39 or 15°C revealed discrete slope discontinuities in plots of polarization vs. temperature. Two well-defined ‘break points’ were present in the 0–40°C temperature range examined and their precise location was dependent upon the growth temperature of the cells. By mixing phospholipids from cells grown at different temperatures, the break points at 17.5 and 32°C in 39°C-lipid multilayer preparations were shown to correlate with the breaks at 12 and 27°C, respectively, in similar preparations from 15°C-grown cells. The discrete break points were also present, but at slightly different characteristic temperatures, in a phosphatidylcholine fraction and a phosphatidylethanolamine plus 2-aminoethylphosphonolipid fraction purified from the phospholipids and in total microsomal lipids (phospholipids plus the sterol-like triterpenoid, tetrahymanol). However, catalytic hydrogenation of the phospholipid fatty acids or mixing the non-hydrogenated phospholipids with increasing proportions of synthetic dipalmitoyl phosphatidylcholine eliminated the break points. We interpret this discontinuous thermotropic response in microsomal lipids as signalling a lipid phase separation of importance in regulating physiological events.