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B J Litman

Publications and source records attributed to B J Litman.

At least 19 recordsLinked to original sources
PubMed1999

Effect of protein hydration on receptor conformation: decreased levels of bound water promote metarhodopsin II formation.

Neutral solutes were used to investigate the effects of osmotic stress both on the ability of rhodopsin to undergo its activating conformation change and on acyl chain packing in the rod outer segment (ROS) disk membrane. The equilibrium concentration of metarhodopsin II (MII), the conformation of photoactivated rhodopsin, which binds and activates transducin, was increased by glycerol, sucrose, and stachyose in a manner which was linear with osmolality. Analysis of this shift in equilibrium in terms of the dependence of ln(Keq) on osmolality revealed that 20 +/- 1 water molecules are released during the MI-to-MII transition at 20 degrees C, and at 35 degrees C 13 +/- 1 waters are released. At 35 degrees C the average time constant for MII formation was increased from 1.20 +/- 0.09 ms to 1.63 +/- 0.09 ms by addition of 1 osmolal sucrose or glycerol. The effect of the neutral solutes on acyl chain packing in the ROS disk membrane was assessed via measurements of the fluorescence lifetime and anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Analysis of the anisotropy decay of DPH in terms of the rotational diffusion model showed that the angular width of the equilibrium orientational distribution of DPH about the membrane normal was progressively narrowed by increased osmolality. The parameter fv, which is proportional to the overlap between the DPH orientational probability distribution and a random orientational distribution, was reduced by the osmolytes in a manner which was linear with osmolality. This study highlights the potentially opposing interplay between the effect of membrane surface hydration on both the lipid bilayer and integral membrane protein structure. Our results further demonstrate that the binding and release of water molecules play an important role in modulating functional conformational changes for integral membrane proteins, as well as for soluble globular proteins.

Animals↗
PubMed1998

Molecular order and dynamics in bilayers consisting of highly polyunsaturated phospholipids.

The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic bilayer structural properties of symmetrically substituted phosphatidylcholines (PCs) with acyl chains containing no, one, four, or six double bonds and mixed-chain phosphatidylcholines with a saturated sn-1 chain and one, four, or six double bonds in the sn-2 chain. Both the Brownian rotational diffusion (BRD) model and the wobble-in-cone model were fit to all differential polarization data, and the descriptions of the data provided by the BRD model were found to be statistically superior. Global analysis of differential polarization data revealed two statistically equivalent solutions. The solution corresponding to a bimodal orientational distribution function, f(theta), was selected based on the effects of temperature on f(theta) and previous measurements on fixed, oriented bilayers. The overall equilibrium acyl chain order in these bilayers was analyzed by comparing the orientational probability distribution for DPH, f(theta) sin theta, with a random orientational distribution. Orientational order decreased and probe dynamics increased in mixed-chain species as the unsaturation of the sn-2 chain was increased. The degree of orientational order dropped dramatically in the dipolyunsaturated species compared with the mixed-chain phosphatidylcholines, which contained a polyunsaturated sn-2 chain. In terms of both orientational order and probe dynamics, the differences between the highly polyunsaturated species and the monounsaturated species were much greater than the differences between the monounsaturated species and a disaturated PC.

Diphenylhexatriene↗
PubMed1998

Effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.

Cholesterol↗
PubMed1996

Primary alcohols modulate the activation of the G protein-coupled receptor rhodopsin by a lipid-mediated mechanism.

The visual pigment rhodopsin is a prototypical member of the G protein-coupled receptor superfamily. In this study, we have investigated the effect of a series of n-alcohols on the formation of metarhodopsin II (MII), the photoactivated conformation of rhodopsin, which binds and activates transducin. When rhodopsin was photolyzed in the presence of several n-alcohols, increased MII formation was observed in the order ethanol > butanol > hexanol, whereas longer chain n-alcohols inhibited MII formation with decanol > octanol. The magnitude of the stimulatory effects was greater in a more highly unsaturated phospholipid. Alcohols, which enhanced MII formation also increased phospholipid acyl chain packing free volume, while those that decreased this bilayer property inhibited MII formation. An apparent discontinuity in the effect of these alcohols results when their potency is calculated in terms of the total aqueous alcohol concentration. In sharp contrast, a continuous variation in their behavior is observed, when their potency is calculated in terms of the amount of alcohol partitioned in the membrane. Our findings strongly support a lipid-mediated mechanism of action for alcohols on rhodopsin and, by analogy, for other G protein-coupled receptors.

Alcohols↗
PubMed1996

A role for phospholipid polyunsaturation in modulating membrane protein function.

Visual transduction is one of the best characterized G protein--coupled signalling systems. In addition, about 50% of the disk membrane phospholipid acyl chains are 22:6n-3, making this system ideal for determining the role of polyunsaturation in modulating membrane-signalling systems. The extent of formation of metarhodopsin II (MII), the G protein--activating photointermediate of rhodopsin, was studied in phospholipid vesicles composed of a variety of phosphatidylcholines, differing in their acyl chain composition at the sn-2 position. The amount of MII formed increased progressively with the level of acyl chain unsaturation at the sn-2 position. The effect of added cholesterol was to reduce the amount of MII formed. The acyl chain packing free volume of the rhodopsin containing lipid vesicles was characterized by a fractional volume parameter fv derived from measurements of the time-resolved fluorescence anisotropy decay of the hydrophobic membrane probe 1,6-diphenyl-1,3,5-hexatriene. The relationship among sn-2 acyl chain unsaturation, cholesterol content, and MII formation is explained on the basis of variation in fv with bilayer lipid composition and a novel model for the packing of phospholipids containing polyenoic acyl chains, such as 22:6n-3.

Fats, Unsaturated↗
PubMed1994

Effect of ethanol on metarhodopsin II formation is potentiated by phospholipid polyunsaturation.

The role of phospholipids in modulating the effect of ethanol on membrane receptor activation was investigated by studying the extent of metarhodopsin II (MII) formation in vesicles formed POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and PDPC (1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine) and in native rod outer segment disk membranes as a function of ethanol concentration. The equilibrium concentration of MII, the G protein-activating form of photoactivated rhodopsin, was found to increase as a function of ethanol concentration in all three bilayers. Phospholipid composition had a marked effect on ethanol potency, with the presence of polyunsaturated phospholipid acyl chains increasing ethanol potency by 40%. The effects of ethanol on lipid acyl chain packing in POPC and PDPC were investigated using frequency domain anisotropy decay measurements of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene. Enhanced formation of MII due to the presence of ethanol was correlated with the effects of ethanol on acyl chain packing properties. These findings support a phospholipid-mediated mechanism for the action of ethanol in modulating integral membrane receptor conformation.

Cell Membrane↗
PubMed1992

Effect of phosphorylation on receptor conformation: the metarhodopsin I in equilibrium with metarhodopsin II equilibrium in multiply phosphorylated rhodopsin.

The superfamily of membrane-bound receptors, which function in signal transduction by activating a guanine nucleotide binding protein or G-protein in response to agonist binding, shares a number of structural and mechanistic properties. Among these similarities is downregulation of functional activity via receptor phosphorylation. In this study, the effects of intermediate levels of phosphorylation (greater than or equal to 4 added phosphates per receptor molecule) on receptor conformational equilibria are examined by comparing the photochemical properties of phosphorylated and unphosphorylated rhodopsins which were incorporated separately into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles. Postflash spectra reflecting the contributions of metarhodopsins I, II, and III (meta I, meta II, and meta III) were obtained from these samples. Deconvolution of appropriate difference spectra allowed a determination of the concentration of the photointermediates of interest. Meta II is the form of photolyzed rhodopsin which binds and activates the visual G-protein (Gt); thus, its relative abundance at equilibrium and temporal stability are important parameters in determining the efficiency of visual signal transduction. The effects of pH and temperature on the meta I in equilibrium with meta II equilibrium constant (Keq) and the rate of decay of meta II to meta III were examined for the reconstituted phosphorylated and unphosphorylated rhodopsin samples. Keq was essentially unaffected by phosphorylation when measured at pH 7.0 and 8.0 and 20 and 37 degrees C. The decay time (lifetime) of meta II----meta III had a value of approximately 4.7 min in both phosphorylated and unphosphorylated samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Role of sn-1-saturated,sn-2-polyunsaturated phospholipids in control of membrane receptor conformational equilibrium: effects of cholesterol and acyl chain unsaturation on the metarhodopsin I in equilibrium with metarhodopsin II equilibrium.

The effect of phospholipid bilayer acyl chain packing free volume on the equilibrium concentration of the form of photolyzed rhodopsin which initiates visual signal transduction, metarhodopsin II (meta II), is examined in reconstituted systems formed from the saturated phospholipid dimyristoylphosphatidylcholine (DMPC) and in the polyunsaturated phospholipid sn-1-palmitoyl-sn-2-arachidonoylphosphatidylcholine (PAPC) with and without 30 mol% cholesterol. The extent of meta II formation is determined from both flash photolysis measurements and rapidly acquired absorbance spectra. Equilibrium and dynamic properties of the lipid bilayer are characterized by the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH). DPH orientational properties are characterized by fv, a parameter which reflects the volume available for probe reorientation in the bilayer, relative to that available in an unhindered, isotropic environment [Straume, M., & Litman, B. J. (1987) Biochemistry 26, 5121-5126]. The metarhodopsin I in equilibrium with meta II equilibrium constant, Keq has a linear relationship with fv for rhodopsin in PAPC vesicles with and without cholesterol as well as for rhodopsin in DMPC vesicles, and these two correlation lines have different slopes. The correlations between Keq and fv in PAPC and DMPC systems are compared with a similar correlation in the native rod outer segment disk membrane and one reported previously in an egg phosphatidylcholine (egg PC) system [Mitchell, D. C., Straume, M., Miller, J. L., & Litman, B. J. (1990) Biochemistry 29, 9143-9149].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Functional equivalence of metarhodopsin II and the Gt-activating form of photolyzed bovine rhodopsin.

Absorption of a photon by the visual pigment rhodopsin leads to the formation of an activated conformational state, denoted rho*, which is capable of activating the visual G-protein, Gt. The bleaching of rhodopsin can be resolved into a series of spectrally distinct photointermediates. Previous studies suggest that the photointermediate metarhodopsin II (meta II, lambda max of 380 nm) corresponds to the physiologically active form rho*. In the studies reported herein, spectral and enzymological data were analyzed and compared so as to evaluate the temporal correspondence between meta II and rho*. This information was obtained by direct observation of the meta II and rho* decay times in parallel experiments utilizing identical preparations of urea-stripped, bovine retinal rod outer segment disk membranes at pH 8.0, 20 degrees C. Postflash spectra were deconvolved to resolve the meta II absorbance at 380 nm, and a decay time for the loss of meta II of 8.2 min (SD = 0.5 min) was obtained from fitting these data to a single-exponential decay process. The diminishing ability of bleached rhodopsin to activate Gt was measured by monitoring the level of catalyzed exchange of Gt-bound GDP for a nonhydrolyzable GTP analogue. Analysis of the decrease in the initial velocity of nucleotide exchange, measured at various postflash incubation times, yielded a rho* decay time of 7.7 min (SD = 0.5 min) when analyzed as a single-exponential process. The similarity of these decay times provides direct evidence that meta II and rho* are present over the same time regime, and further supports the equivalence of these two forms of photoactivated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Packing characteristics of highly unsaturated bilayer lipids: Raman spectroscopic studies of multilamellar phosphatidylcholine dispersions.

The thermotropic properties and acyl chain packing characteristics of multilamellar dispersions of highly unsaturated lipids were examined by Raman spectroscopy. Bilayer assemblies were composed of POPC (1-palmitoyl-2-oleoylphosphatidylcholine), PAPC (1-palmitoyl-2-arachidonylphosphatidylcholine), and PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine), lipid systems possessing saturated sn-1 chains and unsaturated sn-2 chains with one, four, and six double bonds, respectively. Raman spectra were recorded in the acyl chain 2800-3100-cm-1 carbon-hydrogen (C-H) stretching and 1100-1200-cm-1 carbon-carbon (C-C) stretching mode regions, spectral intervals reflecting both the inter- and intrachain order/disorder properties of the various lipid dispersions. In order to obtain C-H stretching mode spectra relevant solely to the sn-1 chains of PAPC and PDPC, liquid-phase spectra of arachidonic and docosahexaenoic acid, respectively, were subtracted from the observed phospholipid spectra. The unsaturated sn-2 chains of PAPC and PDPC undergo minimal conformational reorganizations as the bilayers pass from the gel to liquid-crystalline phases. Phase transition temperatures, Tm, derived from statistically fitting the temperature-dependent Raman spectral data are approximately -2.5, -22.5, and -3 degrees C for POPC, PAPC, and PDPC, respectively. As the degree of unsaturation increases from POPC to PAPC and PDPC, the cooperativity of the phase transition, as measured by its breadth, decreases. Estimates of the transition widths from the temperature profiles are approximately 15 degrees C for PAPC and 20 degrees C for PDPC. The behavior of various Raman spectral parameters for the lipid gel phase reflects the formation of lateral microdomains, or clusters, whose packing properties maximize the van der Waals interactions between sn-1 chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallography↗
PubMed1991

Rhodopsin in dimyristoylphosphatidylcholine-reconstituted bilayers forms metarhodopsin II and activates Gt.

The photochemical intermediate metarhodopsin II (meta II; lambda max = 380 nm) is generally identified with rho*, the conformation of photolyzed rhodopsin which binds and activates the visual G-protein, Gt [Emeis, D., & Hoffman, K.P. (1981) FEBS Lett. 136, 201-207]. Purified bovine rhodopsin was incorporated into vesicles consisting of dimyristoylphosphatidylcholine (DMPC), and the rapid formation of a photochemical intermediate absorbing maximally at 380 nm was quantified via both flash photolysis and equilibrium spectral measurements. Kinetic and equilibrium spectral measurements performed above the Tm of DMPC showed that Gt, in the absence of GTP, enhances the production of the 380-nm-absorbing species while reducing the concentration of the 478-nm-absorbing species, metarhodopsin I (meta I), in a manner similar to that observed in the native rod outer segment disk membrane. This Gt-induced shift in the equilibrium concentration of photointermediates indicated that the species with an absorbance maximum at 380 nm was meta II. The presence of rho* in the DMPC bilayer was established via measurements of photolysis-induced exchange of tritiated GMPPNP, a nonhydrolyzable analogue of GTP, on Gt. Above Tm, the metarhodopsin equilibrium is strongly shifted toward meta I relative to the native rod outer segment disk membrane; however, at 37 degrees C, 40% of the photointermediates are in the form of meta II. The formation of meta II above Tm is slowed by a factor of ca. 2 relative to the disk membrane. Below Tm, the equilibrium is shifted still further toward meta I, and meta II forms ca. 7 times slower than in the disk membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Interconversion of metarhodopsins I and II: a branched photointermediate decay model.

Flash photolysis experiments designed to monitor the establishment of the metarhodopsin I to metarhodopsin II equilibrium are interpreted according to a branched model in which two spectrally indistinguishable but kinetically distinguishable forms of metarhodopsin II are postulated to exist in equilibrium with a common pool of metarhodopsin I. This interpretation arises from the consistent requirement for at least three exponentials for a valid description of the observed growth of absorbance at 380 nm following bleaching of bovine rhodopsin in rod outer segment disk membranes. Analysis of the 380-nm transient absorbance data permitted direct determination of the five physically interpretable individual rate constants of the model. This analysis represents a more explicit interpretation of kinetic data than that employed in earlier experiments of this kind, which involved estimating only apparent rates and apparent amplitudes of discrete multiexponential functions. The 380-nm absorbance contributions of all relevant species contributing to the observed dynamic absorbance change were accounted for simultaneously during nonlinear least-squares estimation of the model rate parameters. Analysis of deconvoluted equilibrium spectra acquired from samples identical with those used in the kinetics experiments confirmed the metarhodopsin I-metarhodopsin II equilibrium constants, Keq, derived from the dynamic analyses. It is shown that Keq varies from 1.28 at 10 degrees C to 7.3 at 37 degrees C and that approximately 90% of the metarhodopsin II present is in the form of metarhodopsin IIslow over the temperature range 10-37 degrees C. A physical interpretation of this decay model is discussed in the context of a distribution of metarhodopsin II structural and energetic states.

Cell Membrane↗
PubMed1990

Modulation of metarhodopsin formation by cholesterol-induced ordering of bilayer lipids.

The effect of lipid ordering on the kinetics and extent of metarhodopsin II (meta II) formation was evaluated in bovine rhodopsin which had been reconstituted into phosphatidylcholine vesicles containing 0, 15, and 30 mol% cholesterol. The rate of establishment of the dynamic equilibrium between metarhodopsin I (meta I) and the two kinetically distinguished forms of meta II in the branched meta II model [meta IIfast and meta IIslow; Straume, M., Mitchell, D. C., Miller, J. L., & Litman, B. J. (1990) Biochemistry (preceding paper in this issue)] is derived from kinetic measurements of rhodopsin photolysis in these vesicle systems at several temperatures. Values of the meta I in equilibrium with meta IItotal equilibrium constant, Keq, are calculated from the derived model-dependent rate constants, and are shown to be equivalent to those derived from rapidly acquired absorbance spectra. The presence of 30 mol% cholesterol reduces Keq by approximately 50% between 10 and 37 degrees C. Analysis of the model-dependent parameters in terms of delta H and delta S reveals that cholesterol raises the free energy of meta IIslow, relative to meta I, by increasing delta H whereas it raises the relative free energy of meta IIfast by making delta S meta IIfast relative to meta I less positive. The reduction in Keq by both temperature and cholesterol is found to be directly correlated with a parameter that reflects the free volume available for molecular motion in the hydrophobic core of the bilayer [Straume, M., & Litman, B. J. (1988) Biochemistry 27, 7723-7733].(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1990

States of aggregation and phase transformations in mixtures of phosphatidylcholine and octyl glucoside.

The result of mixing varying concentrations of the nonionic detergent octyl glucoside (OG) with small unilamellar vesicles (SUV) of egg phosphatidylcholine (PC) made by sonication depends on the ratio between OG and PC in the mixed aggregates. When this molar ratio (Re) is lower than 1.4, the detergent partitions between the PC vesicles and the aqueous medium with a partition coefficient of K = 0.033 mM-1. As a consequence of introduction of OG into the bilayers, the vesicles grow in size. The resultant vesicles have a mean diameter that is an increasing function of Re and is independent of the total PC concentration. Experiments in which the vesicles were loaded with high molecular weight dextran prior to being exposed to OG suggest that the mechanism responsible for the size growth involves lipid transfer rather than fusion. Mixtures with Re values within the range of 1.4-3.2 separate into two macroscopic phases: The lower phase is clear but very viscous. It contains constant OG and PC concentrations and is characterized by an Re value of 3.2, independent of the composition of the whole dispersion. The upper phase contains vesicles of varying concentrations of OG and PC, but a constant Re of 1.4. When the saturating level of 1.4 OG molecules per PC molecule is approached, the concentration of OG monomers in the aqueous medium reaches the value of 16.6 +/- 0.3 mM, which is the apparent cmc of OG in the lipid-containing medium. OG-PC mixed micelles contain at least 3.2 OG molecules per PC molecule. The mixed micelles present at Re = 3.2 apparently have the shape of oblate ellipsoids with a minor axis of about 2 nm and two major axes of about 25 nm. The surface area of the mixed micelles at this point is just sufficient for them to undergo conversion into the smallest possible spherical vesicles of a radius of 12 nm. At Re values above 3.2, the major axis of the mixed micelles becomes smaller as Re increases, while at values of Re below 3.2 the micelles would have been expected to grow very rapidly with decreasing Re. This may explain the partial vesicle closure occurring below Re = 3.2.

Detergents↗
PubMed1989

Inhibition of transducin activation and guanosine triphosphatase activity by aluminum ion.

Aluminum ion perturbs the activity of a number of physiologically important enzymes, including members of a family of guanine nucleotide-binding proteins (G-proteins). G-proteins couple cellular receptor proteins to a variety of effector enzymes (including adenylate cyclase, phospholipase C, and the rod photoreceptor phosphodiesterase). We show herein that subnanomolar concentrations of free aluminum ion, produced in a carefully defined and kinetically stable manner through the buffering of total aluminum at 0.1-1.0 mM with calculated ratios of chelating agents, inhibit both the receptor-mediated activation and the self-inactivating GTPase activity of the rod photoreceptor G-protein, Gv. In the presence of 4 X 10(-10) M free aluminum ion, GTPase activity is inhibited from about 25-60% as the magnesium ion concentration is reduced from 10(-3) to about 5 X 10(-5) M. The principal effect of aluminum ion upon Gv is to inhibit receptor catalyzed nucleotide exchange. Binding of the GTP analog 5'-guanylyl imidodiphosphate can be reduced by as much as 90% by aluminum ion following subsaturating rhodopsin stimulation. Aluminum ion can produce either competitive or mixed noncompetitive inhibition of rhodopsin-catalyzed Gv activation and GTPase activity, as a function of whether Gv undergoes single (competitive), or multiple (mixed noncompetitive) nucleotide exchanges. The rod photoreceptor phosphodiesterase is only slightly inhibited by similar aluminum ion activities. Light- and Gv-coupled phosphodiesterase activation exhibits both a lower maximum rate of cyclic guanosine monophosphate hydrolysis and a slower inactivation in the presence of aluminum ion activities from about 10(-12) - 10(-10) M. These data suggest that intracellular free aluminum ion concentrations in the subnanomolar range could markedly affect the ability of cells to transduce extracellular signals. Interestingly, the combination of Al3+ and F- to produce the fluoro-aluminate species (AlFx) also inhibits the GTPase of G-proteins, although the mechanism of inhibition (e.g. binding to the G-protein.Mg2+.GDP complex) is totally distinct from that observed for free Al3+ and the overall effect on signal transduction (e.g. enhanced signal amplification) is in complete opposition to that observed for free Al3+.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1988

Equilibrium and dynamic bilayer structural properties of unsaturated acyl chain phosphatidylcholine-cholesterol-rhodopsin recombinant vesicles and rod outer segment disk membranes as determined from higher order analysis of fluorescence anisotropy decay.

Limited-frequency phase-modulation fluorometry of diphenylhexatriene (DPH) and trimethylammonium-diphenylhexatriene (TMA-DPH) was used to characterize the equilibrium and dynamic lipid structural properties of (1) reconstituted egg phosphatidylcholine (egg PC)-rhodopsin vesicles varying in rhodopsin content from 0 to approximately 1 mol %, (2) reconstituted PC-cholesterol-rhodopsin vesicles containing approximately 1 mol % rhodopsin and 0, approximately 15, or approximately 30 mol % cholesterol with egg PC, DOPC (di-18:1-PC), or PAPC (16:0,20:4-PC) as the phospholipid constituent, and (3) native bovine rod outer segment disk membranes. Experiments were conducted at 37, 25, 15, and 5 degrees C. Fluorescence lifetime analysis was performed by fitting the data to a constrained, discrete, biexponential model. Rotational depolarization properties were considered by a model requiring a single rotational diffusion coefficient and capable of producing orthogonal, bimodal orientational distributions for DPH and unimodal distributions for TMA-DPH [Straume, M., & Litman, B. J. (1987) Biochemistry 26, 5113-5120]. Unbleached rhodopsin reduced mean fluorophore lifetimes in proportion to the amount of protein present in PC vesicles as a result of probe-to-retinal energy transfer by (1) redistributing the relative lifetime contributions in favor of the short lifetime population and (2) reducing the lifetimes of each derived population. Lifetimes were increased by cholesterol and by reduction of the temperature, but the relative proportions of derived short- and long-lifetime populations were not affected. TMA-DPH lifetimes were more sensitive (in a relative manner) than were those of DPH. These observations are interpreted in terms of cholesterol and reduced temperature each inhibiting water penetrability into these bilayers, with a greater effect occurring in the headgroup and interfacial regions (probed by TMA-DPH) than in the hydrophobic bilayer interior (probed by DPH). Diunsaturated DOPC-rhodopsin recombinants were more resistant to temperature-dependent lifetime changes than were mixed-chain egg PC or PAPC vesicles. This suggests less favorable interaction of rhodopsin with diunsaturated PCs than with mixed-chain PCs. Lifetimes in disk membranes exhibited this same temperature dependence although DPH in disks had lifetimes longer than those seen in recombinant vesicles. TMA-DPH lifetimes in disks were more similar to those observed in cholesterol-containing recombinants. It would therefore appear that the large proportion of small, charged (at pH 7) phosphatidylethanolamine and phosphatidylserine headgroups present in disks reduces water penetrability into the d

Animals↗