Structural effects of substrate utilization on the adenosinetriphosphatase chains of sarcoplasmic reticulum

Addition of ATP to suspensions of fragmented sarcoplasmic reticulum (SR) containing low concentrations of a detergent that does not by itself produce major vesicular disruption is followed by a transient reduction in turbidity accompanied by solubilization of the vesicles. The effect of ATP is Ca2+ dependent and proceeds in parallel with utilization of the nucleotide as a substrate for the SR ATPase. Analogous effects are observed with other substrates producing enzyme phosphorylation at the catalytic site. The effect of ATP can also be detected in studies of fluorescence energy transfer between enzyme chains, by using the technique of Vanderkooi et al. [Vanderkooi, J., Ierokomas, A., Nakamura, H., & Martonosi, A. (1977) Biochemistry 16, 1262]. For this purpose, ATPase chains are labeled separately with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) and 6-(iodoacetamido) fluorescein (IAF). Samples of vesicles uniformly labeled with either IAEDANS or IAF, mixtures of two populations of vesicles uniformly labeled with either fluorophore, and vesicles containing randomized chains labeled with either fluorophore are used as experimental systems. In the last system, significant energy transfer from IAEDANS (donor) to IAF (acceptor) is revealed by fluorescence spectra and measurements of donor fluorescence intensity and lifetime. This is attributed to close interactions between ATPase chains within the membrane bilayer. It is then found that in the presence of low detergent concentrations, ATP changes the extent of energy transfer between labeled ATPase chains, consistent with destabilization of the interaction of chains. The observed effects are attributed to a reversible structural transition concomitant with enzyme phosphorylation and related to catalytic and transport function.     

Resonance energy transfer study of membrane-bound aggregates of the sarcoplasmic reticulum calcium ATPase

The aggregation of the membrane-bound calcium ATPase from sarcoplasmic reticulum has been studied by resonance energy transfer. The temperature dependence of resonance energy transfer from a fluorescent membrane lipid donor to an acceptor covalently linked to the Ca2+ ATPase was observed for the native sarcoplasmic reticulum vesicles and for purified protein reconstituted into phospholipid vesicles. The efficiency of energy transfer in these systems increases as the size of protein aggregates decrease. This is due to the increased exposure of the protein in the lipid domain that results in the shortening of distances between donors and acceptors. The degree of aggregation was observed to decrease with increasing temperature. Aggregates rea h a limiting size at low temperature (5 degrees C) but not a high temperatures (45 degrees C). For the reconstituted system, the aggregate size showed a continuous, smooth decrease with increasing temperature. Sarcoplasmic reticulum vesicles showed a decrease in aggregation except for a region from 20 to 30 degrees C in which no change occurred. Arrhenius plots of the calcium transport activities for both systems do not reflect these differences, but instead show similar discontinuities and activation energies. A theoretical model is used to analyze the resonance energy transfer results for the reconstituted vesicles. The average radius of the ATPase aggregate is obtained from this analysis. The limiting, low temperature value of the aggregate radius is consistent with the formation of a tetramer. This structure breaks down to smaller, functional units at higher temperatures.     

Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum.

Ca2+-ATPase molecules were labeled in intact sarcoplasmic reticulum (SR) vesicles, sequentially with a donor fluorophore, fluorescein-5'-isothiocyanate (FITC), and with an acceptor fluorophore, eosin-5'-isothiocyanate (EITC), each at a mole ratio of 0.25-0.5 mol/mol of ATPase. The resonance energy transfer was determined from the effect of acceptor on the intensity and lifetime of donor fluorescence. Due to structural similarities, the two dyes compete for the same site(s) on the Ca2+-ATPase, and under optimal conditions each ATPase molecule is labeled either with donor or acceptor fluorophore, but not with both. There is only slight labeling of phospholipids and other proteins in SR, even at concentrations of FITC or EITC higher than those used in the reported experiments. Efficient energy transfer was observed from the covalently bound FITC to EITC that is assumed to reflect interaction between ATPase molecules. Protein denaturing agents (8 M urea and 4 M guanidine) or nonsolubilizing concentrations of detergents (C12E8 or lysolecithin) abolish the energy transfer. These results are consistent with earlier observations that a large portion of the Ca2+-ATPase is present in oligomeric form in the native membrane. The technique is suitable for kinetic analysis of the effect of various treatments on the monomer-oligomer equilibrium of Ca2+-ATPase. A drawback of the method is that the labeled ATPase, although it retains conformational responses, is enzymatically inactive.     

Interaction between the Ca2(+)-ATPase and the proteolipid in artificial membranes

The interaction between the Ca2+ transport ATPase and the proteolipid of rabbit sarcoplasmic reticulum was analyzed by fluorescence energy transfer, using the following donor: acceptor combinations: Ca2(+)-ATPase tryptophan----IAEDANS-proteolipid; IAEDANS-ATPase----IAF-proteolipid; IAEDANS-proteolipid----IAF-ATPase. The observed energy transfer may indicate weak interaction between the Ca2(+)-ATPase and proteolipid, but collisional energy transfer definitely contributes. The energy transfer was abolished by deoxycholate or sodium dodecylsulfate at concentrations sufficient to solubilize the membrane. In view of the low proteolipid content of sarcoplasmic reticulum and the weak interaction suggested by the energy transfer, at best only a small fraction of ATPase molecules could exist in the form of ATPase-proteolipid complexes.     

Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles.

Unconjugated bilirubin (bilirubin-IX alpha), the hydrophobic end product of heme degradation, is esterified in the hepatocyte endoplasmic reticulum to water-soluble conjugates prior to excretion in bile. To characterize the process of intracellular bilirubin transport, the kinetic and thermodynamic activation parameters for the spontaneous transfer of bilirubin between small unilamellar egg lecithin vesicles were determined. Bilirubin-IX alpha was added to donor vesicles labeled with the fluorescent phospholipid probe, (5-(dimethylamino)naphthalene-1-sulfonyl) dipalmitoyl-L-alpha-phosphatidylethanolamine (dansyl-PE). When bound to the donor vesicles, bilirubin quenches the dansyl probe fluorescence through resonance energy transfer. The movement of bilirubin from dansyl-labeled donor vesicles to unlabeled acceptor vesicles was monitored directly by the reemergence of dansyl fluorescence over time. Vesicle fusion and intervesicle transfer of the dansyl-PE probe were excluded by quasielastic light scattering and fluorescence resonance energy transfer studies. Stopped-flow analysis demonstrated that the transfer of bilirubin was described by a single-exponential function with a mean half-time of 2.0 +/- 0.1 ms (+/- SD) at 37 degrees C. The rate of bilirubin transfer was independent of acceptor vesicle concentration and decreased with increasing buffer ionic strength, indicating that intermembrane transfer occurred via aqueous diffusion, rather than vesicle collisions. The free energy of activation (delta G++) for the dissociation of bilirubin from donor vesicles was 14.2 kcal.mol-1. These studies suggest that bilirubin is associated with phospholipid bilayers at the membrane-water interface. We postulate that the movement of unconjugated bilirubin between intracellular membranes occurs via spontaneous transfer through the aqueous phase.     

Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum

The interaction between Ca2+-ATPase molecules in the native sarcoplasmic reticulum membrane and in detergent solutions was analyzed by chemical crosslinking, high performance liquid chromatography (HPLC), and by the polarization of fluorescence of fluorescein 5'-isothiocyanate (FITC) covalently attached to the Ca2+-ATPase. Reaction of sarcoplasmic reticulum vesicles with glutaraldehyde causes the crosslinking of Ca2+-ATPase molecules with the formation of dimers, tetramers and higher oligomers. At moderate concentrations of glutaraldehyde solubilization of sarcoplasmic reticulum by C12 E8 or Brij 36T (approximately equal to 4 mg/mg protein) decreased the formation of higher oligomers without significant interference with the appearance of crosslinked ATPase dimers. These observations are consistent with the existence of Ca2+-ATPase dimers in detergent-solubilized sarcoplasmic reticulum. Ca2+ (2-20 mM) and glycerol (10-20%) increased the degree of crosslinking at pH 6.0 both in vesicular and in solubilized sarcoplasmic reticulum, presumably by promoting interactions between ATPase molecules; at pH 7.5 the effect of Ca2+ was less pronounced. In agreement with these observations, high performance liquid chromatography of sarcoplasmic reticulum proteins solubilized by Brij 36T or C12 E10 revealed the presence of components with the expected elution characteristics of Ca2+-ATPase oligomers. The polarization of fluorescence of FITC covalently attached to the Ca2+-ATPase is low in the native sarcoplasmic reticulum due to energy transfer, consistent with the existence of ATPase oligomers (Highsmith, S. and Cohen, J.A. (1987) Biochemistry 26, 154-161); upon solubilization of the sarcoplasmic reticulum by detergents, the polarization of fluorescence increased due to dissociation of ATPase oligomers. Based on its effects on the fluorescence of FITC-ATPase, Ca2+ promoted the interaction between ATPase molecules, both in the native membrane and in detergent solutions.     

Effect of the purified (Mg2+ + Ca2+)-activated ATPase of sarcoplasmic reticulum upon the passive Ca2+ permeability and ultrastructure of phospholipid vesicles.

The passive Ca2+ permeability of fragmented sarcoplasmic reticulum membranes is 10(4) to 10(61 times greater than that of liposomes prepared from natural or synthetic phospholipids. The contribution of membrane proteins to the Ca2+ permeability was studied by incorporating the purified [Ca2+ + Mg2+]-activated ATPase into bilayer membranes prepared from different phospholipids. The incorporation of the Ca2+ transport ATPase into the lipid phase increased its Ca2+ permeability to levels approaching that of sarcoplasmic reticulum membranes. The permeability change may arise from a reordering of the structure of the lipid phase in the environment of the protein or could represent a specific property of the protein itself. The calcium-binding protein of sarcoplasmic reticulum did not produce a similar effect. The increased rate of Ca2+ release from reconstituted ATPase vesicles is not a carrier-mediated process as indicated by the linear dependence of the Ca2+ efflux upon the gradient of Ca2+ concentration and by the absence of competition and countertransport between Ca2+ and other divalent metal ions. The increased Ca2+ permeability upon incorporation of the transport ATPase into the lipid phase is accompanied by similar increase in the permeability of the vesicles for sucrose, Na+, choline, and SO42- indicating that the transport ATPase does not act as a specific Ca2+ channel. Native sarcoplasmic reticulum membranes are asymmetric structures and the 75-A particles seen by freeze-etch electron microscopy are located primarily in the outer fracture face. In reconstituted ATPase vesicles the distribution of the particles between the two fracture faces is even, indicating that complete structural reconstitution was not achieved. The Ca2+ transport activity of reconstituted ATPase vesicles is also much less than that of fragmented sarcoplasmic reticulum. The density of the 40-A surface particles visible after negative staining of native or reconstituted vesicles is greater than that of the intramembranous particles and the relationship between these two structures remains to be established.     

Fluorescence energy transfer between subfragment-1 and actin points in the rigor complex of actosubfragment-1.

The fast-reacting thiol (SH1) of myosin subfragment-1 (S-1) was covalently and specifically labeled with (iodoacetamido)fluorescein (IAF), while Cys-373 of actin was also covalently and preferentially labeled with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (1,5-IAEDANS). The method of fluorescence energy transfer was used to examine the spatial proximity between the two sites, i.e., SH1 and Cys-373, in the rigor complex of acto-S-1. Approximately 30% fluorescence energy transfer was observed from the 1,5-IAEDANS on actin as a donor to the IAF on S-1 as an acceptor in their rigor complex; under certain assumptions this corresponds to a distance of ca. 6.0 nm.     

Interaction of intestinal brush border membrane vesicles with small unilamellar phospholipid vesicles. Exchange of lipids between membranes is mediated by collisional contact

The kinetics of lipid transfer from small unilamellar vesicles as the donor to brush border vesicles as the acceptor have been investigated by following the transfer of radiolabeled or spin-labeled lipid molecules in the absence of exchange protein. The labeled lipid molecules studied were various radiolabeled and spin-labeled phosphatidylcholines, radiolabeled cholesteryl oleate, and a spin-labeled cholestane. At a given temperature and brush border vesicle concentration similar pseudo-first-order rate constants (half-lifetimes) were observed for different lipid labels used. The lipid transfer is shown to be an exchange reaction leading to an equal distribution of label in donor and acceptor vesicles at equilibrium (time t----infinity). The lipid exchange is a second-order reaction with rate constants being directly proportional to the brush border vesicle concentration. The results are only consistent with a collision-induced exchange of lipid molecules between small unilamellar phospholipid vesicles and brush border vesicles. Other mechanisms such as collision-induced fusion or diffusion of lipid monomers through the aqueous phase are negligible at least under our experimental conditions.     

Properties of a reconstituted calcium pump.

1. The translocation of 45Ca2+ in vesicles reconstituted with purified Ca2+ ATPase of sarcoplasmic reticulum and phospholipids was dependent on ATP and varied greatly with the composition of the phospholipids. 2. In contrast to sarcoplasmic reticulum fragments, the reconstituted vesicles were impermeable to 14C-labeled oxalate, 3H- or 32P-labeled ATP, or 32P-i. There was no translocation of phosphate from gamma-labeled ATP during Ca2+ uptake. These results are inconsistent with some current formulations of the mechanism of pump action. 3. Reversal of the Ca2+ pump and generation of ATP and ADP and P-i was observed when vesicles loaded with Ca2+ were exposed to ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. 4. Experiments on the formation of phosphoenzyme with 32P-labeled ATP showed that most if not all functional ATPase molecules in the reconstituted vesicles were oriented in the same direction, as in the case of sarcoplasmic reticulum fragments.     

Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver

Trafficking and sorting of lipids during transport from the endoplasmic reticulum to the Golgi apparatus was studied using a cell-free system from rat liver. Transitional elements of the endoplasmic reticulum were prepared from liver slices prelabeled with [14C]- or [3H]acetate as the donor fraction. Non-radioactive Golgi apparatus were immobilized on nitrocellulose as the acceptor. When reconstituted, the radiolabeled donor retained a capacity to transfer labeled lipids to the non-radioactive Golgi apparatus acceptor. Transfer exhibited two kinetically different components. One was stimulated by ATP, facilitated by cytosol and inhibited by guanosine 5'-O-(thiotriphosphate) and N-ethylmaleimide. In parallel with protein transport, the ATP-dependent lipid transfer occurred with a temperature transition at about 20 degrees C. The other was not stimulated by ATP, did not require cytosol, was acceptor unspecific, was unaffected by inhibitors and, while temperature dependent, did not exhibit a sharp temperature transition. The ATP-independent transfer was non-vesicular. In contrast, the ATP-dependent transfer was vesicular. Transition vesicles isolated by preparative free-flow electrophoresis, when used as the donor fraction, transferred lipids to Golgi apparatus acceptor with a 5-6-fold greater efficiency than that exhibited by the unfractionated transitional endoplasmic reticulum. Formation of transition vesicles was ATP-dependent. Transferred lipids were chiefly phosphatidylcholine and cholesterol. Membrane triglycerides, major constituents of the transitional endoplasmic reticulum membranes, were both depleted in the transition vesicle-enriched fractions and not transferred to Golgi apparatus suggestive of lipid sorting prior to or during transition vesicle formation. The characteristics of the ATP plus cytosol-dependent transfer were similar to those for protein transfer mediated by transition vesicles. Thus, the 50-70-nm vesicles derived from transitional endoplasmic reticulum appear to function in the trafficking of both newly synthesized proteins and lipids from the endoplasmic reticulum to the Golgi apparatus.     

Interactions of hexachlorocyclohexanes with the (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum

Hexachlorocyclohexanes have been shown to inhibit the (Ca2+ + Mg2+)-ATPase of muscle sarcoplasmic reticulum reconstituted into bilayers of dioleoylphosphatidylcholine. However, for the ATPase reconstituted into bilayers of dimyristoleoylphosphatidylcholine, a pattern of activation at low concentration followed by inhibition at higher concentration is seen for hexachlorocyclohexanes and alkanes such as decane and hexadecane. The ATPase in sarcoplasmic reticulum vesicles is also inhibited by the hexachlorocyclohexanes. The effects of hexachlorocyclohexanes on activity are largely independent of concentrations of Ca2+ and ATP. Inhibition is more marked at lower temperatures. The hexachlorocyclohexanes quench the tryptophan fluorescence of the ATPase, and the quenching can be used to obtain partition coefficients into the membrane system. As for simple lipid bilayers, partition exhibits a negative temperature coefficient. Binding is related to effects on ATPase activity.     

Fluorescence coupling across lipid bilayer membranes

Summary An energy transfer between donor and acceptor fluorophores across single lipid bilayer membranes is demonstrated. Anilino-naphthalene sulfonate is used as the donor chromophore: its fluorescence is enhanced by the presence of lipid and thus indicates association with the purely lipid membranes of our preparation of vesicles in suspension. Light emit ted by the donor molecules excites fluorescence of acriflavine, a suitable acceptor enclosed inside the vesicles. Absorption and fluorescence spectra of this system, in its components and as a whole, are presented in evidence for an energy transfer.Supported by a grant from the Medical Research Council of Canada. The results of this work were presented, in part, at the 17th Annual Meeting of the Biophysical Society, February 27–March 2, 1973, Columbus, Ohio.Scholar of the Medical Research Council of Canada.     

Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells

A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) or fluorescein as the energy donor and sulforhodamine as the energy acceptor. The necessary parameters for RET in this system were first optimized using liposomes. Both quenching of the energy donor and sensitized fluorescence of the energy acceptor could be directly observed in the microscope. RET microscopy was then used in cultured fibroblasts to identify those intracellular organelles labeled by the lipid probe, N-SRh-decylamine (N-SRh-C10). This was done by observing the sites of RET in cells doubly labeled with N-SRh-C10 and an NBD-labeled lipid previously shown to label the endoplasmic reticulum, mitochondria, and nuclear envelope. RET microscopy was also used in cells treated with fluorescein-labeled Lens culinaris agglutinin and a sulforhodamine derivative of phosphatidylcholine to examine the internalization of plasma membrane lipid and protein probes. After internalization, the fluorescent lectin resided in most, but not all of the intracellular compartments labeled by the fluorescent lipid, suggesting sorting of the membrane-bound lectin into a subset of internal compartments. We conclude that RET microscopy can co-localize different membrane-bound components at high resolution, and may be particularly useful in examining temporal and spatial changes in the distribution of fluorescent molecules in membranes of the living cell.     

A fluorescence energy transfer method for analyzing protein oligomeric structure: application to phospholamban

We have developed a method using fluorescence energy transfer (FET) to analyze protein oligomeric structure. Two populations of a protein are labeled with fluorescent donor and acceptor, respectively, then mixed at a defined donor/acceptor ratio. A theoretical simulation, assuming random mixing and association among protein subunits in a ring-shaped homo-oligomer, was used to determine the dependence of FET on the number of subunits, the distance between labeled sites on different subunits, and the fraction of subunits remaining monomeric. By measuring FET as a function of the donor/acceptor ratio, the above parameters of the oligomeric structure can be resolved over a substantial range of their values. We used this approach to investigate the oligomeric structure of phospholamban (PLB), a 52-amino acid protein in cardiac sarcoplasmic reticulum (SR). Phosphorylation of PLB regulates the SR Ca-ATPase. Because PLB exists primarily as a homopentamer on sodium dodecyl sulfate polyacrylamide gel electrophoresis, it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, this hypothesis must be tested by determining directly the oligomeric structure of PLB in the lipid membrane. To accomplish this goal, PLB was labeled at Lys-3 in the cytoplasmic domain, with two different amine-reactive donor/acceptor pairs, which gave very similar FET results. In detergent solutions, FET was not observed unless the sample was first boiled to facilitate subunit mixing. In lipid bilayers, FET was observed at 25 degrees C without boiling, indicating a dynamic equilibrium among PLB subunits in the membrane. Analysis of the FET data indicated that the dye-labeled PLB is predominantly in oligomers having at least 8 subunits, that 7-23% of the PLB subunits are monomeric, and that the distance between dyes on adjacent PLB subunits is about 10 A. A point mutation of PLB (L37A) that runs as monomer on SDS-PAGE showed no energy transfer, confirming its monomeric state in the membrane. We conclude that FET is a powerful approach for analyzing the oligomeric structure of PLB, and this method is applicable to other oligomeric proteins.     

The effect of calcium ion transport ATPase upon the passive calcium ion permeability of phospholipid vesicles.

The uptake and release of Ca2+ by sarcoplasmic reticulum fragments and reconstituted ATPase vesicles was measured by a stopped-flow fluorescence method using chlortetracycline as Ca2+ indicator. Incorporation of the Ca2+ transport ATPase into phospholipid bilayers of widely different fatty acid composition increases their passive permeability to Ca2+ by several orders of magnitude. Therefore in addition to participating in active Ca2+ transport, the (Mg2+ + Ca2+)-activated ATPase also forms hydrophilic channels across the membrane. The relative insensitivity of the permeability effect of ATPase to changes in the fatty acid composition of the membrane is in accord with the suggestion that the Ca2+ channels arise by protein-protein interaction between four ATPase molecules. The reversible formation of these channels may have physiological significance in the rapid Ca2+ release from the sarcoplasmic reticulum during activation of muscle.     

Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer.

We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 A in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 A for NBD and 31-55 A for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.     

Intermonomer flexibility of Ca- and Mg-actin filaments at different pH values.

The fluorescence resonance energy transfer parameter, f, is defined as the efficiency of the energy transfer normalized by the quantum yield of the donor in the presence of acceptor. It is possible to characterize the flexibility of the protein matrix between the appropriate fluorescent probes by monitoring the temperature dependence of f. The intermonomer flexibility of the Ca-actin and Mg-actin filaments was characterized by using this method at pH values of 6.5 and 7.4. The protomers were labeled on Cys374 with donor [N-(((iodoacetyl)amino)ethyl)-5-naphthylamine-1-sulfonate; IAEDANS] or acceptor [5-(iodoacetamido)fluorescein; IAF] molecules. The temperature profile of f suggested that the intermonomer flexibility of actin filaments was larger at pH 7.4 than pH 6.5 in the case of Mg-F-actin while this difference was absent in the case of Ca-F-actin. More rigid intermonomer connection was identified at both pH values between the protomers of Mg-F-actin compared to the Ca-F-actin. The results were further supported by time dependent fluorescence measurements made on IAEDANS and IAF labeled Mg- and Ca-actin filaments at pH 6.5 and 7.4. Our spectroscopic results may suggest that the altered function of muscle following the change of pH within the muscle cells under physiological or pathological conditions might be affected by the modified dynamic properties of the magnesium saturated actin filaments. The change of the intracellular pH does not have an effect on the intermonomer flexibility of the Ca-actin filaments.     

Aggregation, lipid exchange, and metastable phases of dimyristoylphosphatidylethanolamine vesicles

A new fluorescent lipid analogue, bimanephosphatidylcholine, has been synthesized for use in lipid bilayers. This probe is well suited as an energy-transfer donor with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine as the acceptor. Dimyristoylphosphatidylethanolamine vesicles are prepared by sonication at pH 9 and characterized by electron microscopy and other methods. Resonance energy transfer between separately labeled donor and acceptor vesicles is monitored during HCl-induced aggregation to determine the kinetics of lipid randomization. Light scattering is also monitored to measure the kinetics of aggregation. The light scattering shows a marked reversal with NaOH while the energy transfer does not, indicating lipid exchange during a reversibly aggregated state; the extent of energy transfer suggests that only lipids in the outer monolayers exchange. The gel to liquid-crystalline phase transition temperature in HCl-treated vesicles is found to be 47 degrees C with diphenylhexatriene. The initial sonicated dispersion does not show a sharp phase transition. In vesicles labeled with both donor and acceptor probes, a small, irreversible increase in energy transfer is obtained upon lowering and then restoring the pH. These results suggest a metastable phase in the sonicated vesicles containing a randomized distribution of lipid and probes within the bilayers; the thermodynamically favored phase, whose formation is triggered by the pH shock, contains domains within which the probe lipids are more highly concentrated.     

Fluorescence energy transfer from diphenylhexatriene to bacteriorhodopsin in lipid vesicles

Fluorescence energy transfer between the donor diphenylhexatriene (DPH) and the acceptor retinal and fluorescence depolarization of DPH are used to test current theories for fluorescence energy transfer in two-dimensional systems and to obtain information on the effect of the intrinsic membrane protein, bacteriorhodopsin, on the order and dynamics of the lipid phase. Increasing the surface concentration of acceptors by raising the protein to lipid ratio leads to a decrease in the mean fluorescence lifetime by up to a factor of four. When the acceptor concentration is reduced at a fixed protein to lipid ratio by photochemical destruction of retinal, the lifetime increases and reaches approximately the value observed in protein-free vesicles when the bleaching is complete. The shape of the decay curve and the dependency of the mean lifetime on the surface concentration of acceptors are in agreement with theoretical predictions for a two-dimensional random distribution of donors and acceptors. From this analysis a distance of closest approach between donors and acceptors of approximately 18 A is obtained, which is close to the effective radius of bacteriorhodopsin (17 A) and consistent with current ideas about the location of retinal in the interior of the protein. In the absence of energy transfer (bleached vesicles), the steady-state fluorescence anisotropy, -r, of DPH is considerably lower than in the corresponding unbleached vesicles, indicating that the effect of energy transfer must be taken into account when interpreting -r in terms of order and dynamics.