Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.     

A fluorescence anisotropy study on the phase behavior of dimyristoylphosphatidylcholine/cholesterol mixtures

The phase behavior of L-alpha-dimyristoylphosphatidylcholine/cholesterol mixtures was studied in multilamellar vesicles by fluorescence polarization of the sterol molecule dehydroergosterol and of the polyene molecule alpha-parinaric acid. In the absence of cholesterol, dehydroergosterol exhibited an increase in polarization as DMPC vesicles were heated through the phase transition. This rise in polarization anisotropy was observed over a 0.6-1.0 degrees C increase in temperature with the midpoint of the phase transition occurring at 23.6 degrees C. Addition of 5 mol% cholesterol completely obliterated this change in polarization anisotropy through the phase transition of DMPC. alpha-Parinaric acid underwent a characteristic decrease in polarization anisotropy through the phase transition of DMPC. The change in anisotropy through the phase transition was over 4-fold greater than the values observed with dehydroergosterol. Vesicles containing 5 mol% cholesterol in the presence of alpha-parinaric acid underwent a decrease in polarization anisotropy that was over 75% of the original decrease in amplitude observed in the absence of any membrane cholesterol. The difference in sensitivity of the two fluorescent probes to the phase transition of DMPC as a function of membrane cholesterol content may be explained by a preferential partitioning of dehydroergosterol (and cholesterol) into a sterol-rich phase at low sterol concentrations. This partitioning allows dehydroergosterol to detect sterol-rich regions in the membrane bilayer.     

Bilayer structure and physical dynamics of the cytochrome b5 dimyristoylphosphatidylcholine interaction.

Cytochrome b5 is a microsomal membrane protein which provides reducing potential to delta 5-, delta 6-, and delta 9-fatty acid desaturases through its interaction with cytochrome b5 reductase. Low angle x-ray diffraction has been used to determine the structure of an asymmetrically reconstituted cytochrome b5:DMPC model membrane system. Differential scanning calorimetry and fluorescence anisotropy studies were performed to examine the bilayer physical dynamics of this reconstituted system. These latter studies allow us to constrain structural models to those which are consistent with physical dynamics data. Additionally, because the nonpolar peptide secondary structure remains unclear, we tested the sensitivity of our model to different nonpolar peptide domain configurations. In this modeling approach, the nonpolar peptide moiety was arranged in the membrane to meet such chemically determined criteria as protease susceptibility of carboxyl- and amino-termini, tyrosine availability for pH titration and tryptophan 109 location, et cetera. In these studies, we have obtained a reconstituted cytochrome b5:DMPC bilayer structure at approximately 6.3 A resolution and conclude that the nonpolar peptide does not penetrate beyond the bilayer midplane. Structural correlations with calorimetry, fluorescence anisotropy and acyl chain packing data suggest that asymmetric cytochrome b5 incorporation into the bilayer increases acyl chain order. Additionally, we suggest that the heme peptide:bilayer interaction facilitates a discreet heme peptide orientation which would be dependent upon phospholipid headgroup composition.     

Reconstitution of membrane proteins: catalysis by cholesterol of insertion of integral membrane proteins into preformed lipid bilayers

The presence of cholesterol in small unilamellar vesicles (ULV) of dimyristoylphosphatidylcholine (DMPC) catalyzes fusion of the vesicles at temperatures below the upper limit for the gel to liquid-crystalline phase transition of the DMPC. The extent to which ULV grow depends on the concentration of cholesterol in the vesicles and on temperature. Maximum growth occurs at 21 degrees C. It decreases as the temperature is lowered below 21 degrees C. Growth does not occur at temperatures above the phase transition. In addition, the presence of cholesterol in ULV of DMPC catalyzes the insertion of integral membrane proteins into the vesicles. Thus, bacteriorhodopsin from Halobacterium halobrium, UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, and cytochrome oxidase from beef heart mitochondria formed stable lipid-protein complexes spontaneously when added to ULV containing cholesterol at temperatures under which these vesicles would fuse. Incorporation of these proteins into the ULV of DMPC did not occur in the absence of cholesterol or in the presence of cholesterol when the temperature of the system was above that for the phase transition. It appears that cholesterol lowers the energy barrier for fusion of ULV of DMPC and for insertion of integral membrane proteins into these bilayers. Studies with bacteriorhodopsin suggest that the energy barrier for insertion of proteins into ULV containing cholesterol is smaller than the energy barrier for fusion of the ULV with each other.     

Effect of cholesterol on interaction of dibucaine with phospholipid vesicles: a fluorescence study

Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoyl phosphatidylcholine (DOPC) containing different mol percents of cholesterol has been studied by fluorescence spectroscopy. Fluorescence measurements on dibucaine in presence of phospholipid vesicles containing various amounts of cholesterol yielded a pattern of variation of wavelength at emission maximum and steady-state anisotropy which indicated that the microenvironment of dibucaine is more polar and flexible in membranes that contain cholesterol than in membranes without cholesterol. Experiments on quenching of fluorescence from membrane-associated dibucaine by potassium iodide showed a marked increase in quenching efficiency as the cholesterol content of the vesicles was increased, demonstrating increased accessibility of the iodide quenchers to dibucaine in the presence of cholesterol, when compared to that in its absence. Total emission intensity decay profiles of dibucaine yielded two lifetime components of approximately 1 ns and approximately 2.8--3.1 ns with mean relative contributions of approximately 25 and approximately 75%, respectively. The mean lifetime in vesicles was 20--30% smaller than in the aqueous medium and showed a moderate variation with cholesterol content. Fluorescence measurements at two different temperatures in DMPC SUVs, one at 33 degrees C, above the phase transition temperature and another at 25 degrees C, around the main phase transition, indicated two different mode of dibucaine localization. At 25 degrees C dibucaine partitioned differentially in presence and absence of cholesterol. However, at 33 degrees C the apparent partition coefficients remained unaltered indicating differences in the microenvironment of dibucaine in presence and absence of cholesterol in the phospholipid membranes.     

Deuterium nuclear magnetic resonance spectroscopic study of the fluorescent probe diphenylhexatriene in model membrane systems

We have investigated the deuterium (2H) nuclear magnetic resonance (NMR) spectra of two 2H-labeled fluorescence probes (trans,trans,trans-1,6-diphenylhexa-1,3,5-trienes, DPHs) incorporated into model lipid bilayer membrane systems at various temperatures. The membranes consisted of multilamellar bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) containing varying concentrations of cholesterol. The conventional one-order parameter approach often used in the analysis of the NMR data of lipid membranes does not explain the observed temperature variations of the spectral features. Consistent with the molecular symmetry, the results have thus been analyzed in terms of an ordering matrix with more than one independent element. The molecular order parameter (SNMR), the order along the long molecular axis, in the pure lipid system varies from 0.49 to 0.26 as the temperature is increased from 25 to 57 degrees C. These values are somewhat larger than the order parameters obtained from fluorescence depolarization (SFLU) on sonicated DMPC vesicles. Such discrepancies probably arise from the looser packing of the sonicated vesicles. Addition of cholesterol to the model membranes causes the order parameter of the probe molecules to increase. At 35 degrees C, SNMR increases from 0.38 (with no cholesterol) to 0.92 (in the presence of 50 mol % cholesterol). These values are about 10% larger than those obtained from fluorescence depolarization studies on sonicated vesicles. The SNMR for DPH are somewhat larger than those obtained in earlier NMR studies of 2H-labeled cholesterol. However, they compare well with those obtained for 2H-labeled DMPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein and lipid structural transitions in cytochrome c oxidase-dimyristoylphosphatidylcholine reconstitutions

The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome c oxidase prevents 99 +/- 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high-sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 degrees C and characterized by an enthalpy change of 550 +/- 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 degrees C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes approximately 10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of factor Va on lipid dynamics in mixed phospholipid vesicles as detected by steady-state and time-resolved fluorescence depolarization of diphenylhexatriene

We have monitored the thermotropic behavior of mixed dimyristoylglycerophosphoserine (Myr2GroPSer)/dimyristoylglycerophosphocholine (Myr2GroPCho) and Myr2GroPSer/dipalmitoylglycerophosphocholine (Pam2GroPCho) vesicles in the presence of blood-clotting factor Va, using 1,6-diphenyl-1,3, 5-hexatriene as a lipid probe. The Ca2+-independent interaction of factor Va with these vesicles caused a small increase (1-2 degrees C) in the phase transition temperature, regardless of whether Myr2GroPChe was the lower or higher-melting component of the mixed vesicles. The major effect of factor Va was to increase the polarization of diphenylhexatriene when the mixed vesicles were in the liquid crystalline phase. The protein did not change the anisotropy in the bilayer gel state. The increase in the polarization value above the transition temperature closely correlated with the amount of phospholipid-bound factor Va, as verified by a direct binding technique. In addition, we found that the affinity of factor Va for Myr2GroPSer/Myr2GroPCho and Myr2GroPSer/Pam2GroPCho greatly increased at temperatures above the transition temperatures. Time-dependent fluorescence anisotropy measurements of diphenylhexatriene embedded in vesicles in the liquid crystalline state give fluorescence decay curves which can best be fitted by two exponential functions with two rotational correlation times and a constant term. Vesicles composed of Myr2GroPSer exhibit more ordering than Myr2GroPCho vesicles. However, the order parameter of mixed vesicles composed of 40% Myr2GroPSer and 60% Myr2GroPCho (mol/mol) approached that of Myr2GroPCho. Factor Va dramatically increased the longer rotational correlation time of diphenylhexatriene embedded in mixed vesicles in the liquid crystalline state from 3.7 ns to about 17 ns. The second rank-order parameter increased only slightly, but the calculated steady-state anisotropy increased by twofold. These results indicate that the acidic phospholipid-dependent binding of factor Va to mixed vesicles has an ordering effect on the acyl chains of the acidic phospholipids in the outer layer, but leaves the bulk of the phospholipids, mainly phosphatidylcholine, unaltered. None of the factor-Va-induced alterations in the anisotropy parameters point to the occurrence of lateral phase separation.     

A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Polyphosphoinositide activation of cholesterol side chain cleavage with purified cytochrome P-450scc

Highly purified beef adrenal cytochrome P-450 specific for cholesterol side chain cleavage (P-450-scc) has been reconstituted with sonicated vesicles containing cholesterol and either dimyristoyl phosphatidylcholine (DMPC) or dioleoyl phosphatidylcholine (DOPC). When cholesterol was present in DMPC vesicles at 1:15 molar ratio, cardiolipin and L-alpha-phosphatidylinositol 4-monophosphate (DPI) increased side chain cleavage by at least 5-fold (0.7 min-1-3.5 min-1). In DOPC vesicles, a smaller increase was observed (2.8 min-1-5.0 min-1). Activator phospholipids increased the rate of transference of cholesterol both to and from the cytochrome when, respectively, cholesterol-free P-450scc and cholesterol-P-450scc complex are combined with either DMPC or DOPC vesicles. Transfer of cholesterol to and from cytochrome P-450 occurred with similar first order rate constants and was also independent of the concentrations of cholesterol vesicles and P-450. It is suggested that transfer in both directions is limited by the rate of insertion of P-450scc into the membrane. Phospholipid stimulatory effects for both cholesterol transfer and for activation of side chain cleavage occurred with the same ranking, even though cholesterol transfer, following reconstitution, was 5-10 times slower than the turnover of side chain cleavage. DPI increased Vmax for side chain cleavage in both DMPC and DOPC vesicles to the same rate (12 min-1) without effect on the Km for cholesterol, while cardiolipin both produced a similar increase in Vmax and decreased Km (cholesterol). This activation by DPI is attributed to more favorable incorporation of P-450scc in these membranes and is consistent with previously reported effects of acidic phospholipids on other mitochondrial proteins.     

Calcium-dependent and calcium-independent interactions of prothrombin fragment 1 with phosphatidylglycerol/phosphatidylcholine unilamellar vesicles

We have measured the phase behavior of mixed dipentadecanoylphosphatidylglycerol (DC15PG)/dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUV) in the presence of saturating (greater than 98% occupancy of binding sites) concentrations of bovine prothrombin fragment 1 and 5 mM Ca2+. Binding of fragment 1 in the presence of Ca2+ was verified by an increase in 90 degrees light scattering. Only in the cases of DC15PG/DMPC SUV below their phase transition and of pure DMPC SUV were such light scattering measurements not reversible upon addition of ethylenediaminetetraacetic acid to complex Ca2+. Phase-behavior changes of DC15PG/DMPC SUV as monitored by diphenylhexatriene fluorescence anisotropy occurred in concert with the binding of fragment 1. The major effects of peptide binding on SUV phase behavior were to raise the phase-transition temperature by 2-15 degrees C, depending on vesicle composition, and, in general, to make the phase diagram for these small vesicles closely resemble that of large vesicles. No evidence was obtained for the existence of lateral membrane domains with distinct compositions induced by the binding of prothrombin fragment 1 plus Ca2+. Surprisingly, fragment 1 without Ca2+ also altered the phase behavior of DC15PG/DMPC SUV. Most striking was the effect of fragment 1 (with or without Ca2+) on DMPC SUV phase behavior. Freeze-fracture electron microscopy demonstrated that pure DMPC vesicles were induced to fuse in the presence of fragment 1, while vesicles containing DC15PG remained intact. The rate of DMPC SUV fusion (followed by 90 degrees light scattering) increased with increasing fragment 1 concentration but was not saturable up to 40 microM fragment 1, suggesting a weak, nonspecific interaction between fragment 1 and the neutral phospholipid vesicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome b5 and NADH-cytochrome-b5 reductase from sipunculan erythrocytes; a methemerythrin reduction system from Phascolopsis gouldii

We report the purification and characterization of a soluble cytochrome b5 from coelomic erythrocytes of the sipunculan worm, Phascolopsis gouldii. We also report the isolation and purification of a membrane-bound NADH-cytochrome-b5 reductase from these erythrocytes. The non-heme iron protein, hemerythrin (Hr), is known to be the oxygen carrier in these erythrocytes. The aforementioned purified cytochrome b5 and reductase together catalyze the reduction of P. gouldii [Fe(III),Fe(III)]metHr to [Fe(II),Fe(II)deoxyHr by NADH. EPR spectroscopy demonstrates that a redox process involving formation of the intermediate [Fe(II),Fe(III)]semi-metHr occurs within intact sipunculan erythrocytes as well as in the system of purified components. The rhombic g-tensor of the EPR signal in both cases resembles that of (semi-met)RHr, the form obtained by one-electron reduction of metHr. These observations suggest that cytochrome b5 and NADH-cytochrome-b5 reductase in sipunculan erythrocytes function to counteract autoxidation of oxyHr. The sequence of electron flow in the system of purified components is: NADH----NADH-cytochrome-b5 reductase----cytochrome b5----metHr. At pH 7.5, the reduction of metHr in this system occurs in two phases, only the first of which is dependent on concentration of cytochrome b5. From an analysis of the kinetics and the EPR time-course, we propose that the two phases represent sequential reduction of met- to semi-metHr and reduction of semi-metHr to deoxyHr. This report represents the first demonstration of a physiological system for reduction of metHr.     

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay.

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.     

Calorimetric studies of cytochrome oxidase-phospholipid interactions

Thermotropic phase transitions in phospholipid vesicles reconstituted with mitochondrial cytochrome oxidase (EC 1.9.3.1) were studied using differential scanning calorimetry. Both dimyristoylphosphatidylcholine (DMPC) and mixtures of DMPC and cardiolipin were used at different lipid-to-protein ratios. The incorporated protein reduces the energy absorbed during phase transitions of DMPC vesicles, and causes a small decrease in the transition temperature (tm). delta H depends on the amount of protein in the vesicles. This dependence indicates that about 72 DMPC molecules are influenced per cytochrome alpha alpha 3 monomer. The transition parameters remain unaffected by changes in ionic strength or by reduction of the enzyme. Incorporation of cytochrome oxidase depleted of subunit III into DMPC liposomes resulted in a larger decrease of tm, but the amount of perturbed phospholipids remains similar to that in the case of the intact enzyme. Incorporation of cytochrome oxidase into DMPC/cardiolipin vesicles counteracts the effect of cardiolipin in decreasing the enthalpy of the DMPC transition. Thus cytochrome oxidase segregates the phospholipids by attracting cardiolipin from the bulk lipid. Cytochrome c does not significantly affect this apparent cardiolipin 'shell' around membranous cytochrome oxidase.     

Nanosecond time-resolved fluorescence investigations of temperature-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicles and solubilized systems

Intrinsic and lipid phase transition-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicle and solubilized systems were examined by the fluorescence lifetime of N-(1-anilinonaphthyl-4)-maleimide conjugated with the enzyme. The time-dependent fluorescence intensity of N-(1-anilinonaphthyl-4)-maleimide attached to cytochrome oxidase was described as a triple exponential decay. Both the intrinsic and lipid phase transition-induced conformational changes were detectable in plots of the average lifetime against temperature. In most cases a peak occurred at the temperature of the conformational change. The time-dependent emission anisotropy showed that N-(1-anilinonaphthyl-4)-maleimide embedded in cytochrome oxidase in phosphatidylcholine vesicles underwent a rapid restricted wobbling within a cone. The half-angle of the cone was around 30 degrees for cytochrome oxidase in dimyristoyl phosphatidylcholine vesicles.     

Effect of proteins on fluorophore lifetime heterogeneity in lipid bilayers.

The effect of three different membrane proteins on the fluorescence lifetime heterogeneity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in phospholipid vesicle systems was investigated. For large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) at 37 degrees C, the fluorescence decay was essentially monoexponential (8.6 and 8.2 ns, respectively) except for a minor component typical of DPH. For gramicidin D reconstituted into DMPC vesicles at a protein/lipid molar ratio of 1/7, the most appropriate analysis of the data was found to be in the form of a bimodal Lorentzian distribution. Centers of the major lifetime components were almost identical with those recovered for vesicles without proteins, while broad distributional widths of some 4.0 ns were recovered. Variation of the protein/lipid molar ratio in sonicated POPC vesicles revealed an abrupt increase in distributional width at ratios approximating 1/15-1/20, which leveled off at about 2.5 ns. For bacteriorhodopsin in DMPC vesicles and cytochrome b5 in POPC, the most appropriate analysis of the data was again found to be in the form of a bimodal Lorentzian also with broad distributional widths in the major component. Lifetime centers were decreased for these proteins due to fluorescence energy transfer to the retinal of the bacteriorhodopsin and heme of the cytochrome b5. Fluorescence energy transfer is distance dependent, and since a range of donor-acceptor distances would be expected in a membrane, lifetime distributions should therefore be recovered independently of other effects for proteins possessing acceptor chromophores.(ABSTRACT TRUNCATED AT 250 WORDS)     

The rotational diffusion of cytochrome b5 in lipid bilayer membranes. Influence of the lipid physical state.

A derivative of the integral membranes protein, cytochrome b5, has been prepared in which the native heme group has been replaced by the structurally similar rhodium(III)-protoporphyrin IX. This metalloporphyrin has a finite triplet yield with a single exponential decay time of 22 microsecond in water. After insertion of the metalloporphyrin into the protein, its triplet-state decay becomes strongly nonexponential with at least three equal amplitude components with time constants varying over a range of 100. The derivatized protein has been incorporated into unilamellar liposomes prepared from dimyristoyllecithin, and the rotational diffusion of the protein in the lipid bilayer has been studied at temperatures above and below the lipid phase transition temperature via triplet absorbance anisotropy decay. The anisotropy decay curves are biphasic both above and below the lipid phase transition. The rotational diffusion constant is found to be 2.4 X 10(5) s-1 at 35 degrees C, and 1.1 X 10(4) s-1 at 10 degrees C, both being calculated from the fast decay component. The ratio of the limiting anisotropy to the initial anisotropy is 0.6 at both temperatures. This implies a cone of restricted motion of 34 degrees for the protein in the bilayer.     

Reduction of cytochrome b5 by NADPH-cytochrome P450 reductase

The reduction of mammalian cytochrome b5 (b5) by NADPH-cytochrome P450 (P450) reductase is involved in a number of biological reactions. The kinetics of the process have received limited consideration previously, and a combination of pre-steady-state (stopped-flow) and steady-state approaches was used to investigate the mechanism of b5 reduction. In the absence of detergent or lipid, a reductase-b5 complex is formed and rearranges slowly to an active form. Electron transfer to b5 is rapid within this complex (>30 s(-1) at 23 degrees C), as fast as to cytochrome c. With excess b5 present, a burst of reduction is observed, consistent with rapid electron transfer to one or two b5 molecules per reductase, followed by a subsequent rate-limiting event. In detergent vesicles, the reductase and b5 interact rapidly but electron transfer is slower (approximately 3 s(-1) at 23 degrees C). Experiments with dimyristyl lecithin vesicles yielded results intermediate between the non-vesicle and detergent systems. These steady-state and pre-steady-state kinetics provide views of the different natures of the reduction of b5 by the reductase in the absence and presence of vesicles. Without vesicles, the encounter of the reductase and b5 is rapid, followed by a slow reorganization of the initial complex (approximately 0.07 s(-1)), very fast reduction, and dissociation. In vesicles, encounter is rapid and the slow step (approximately 3 s(-1)) is reduction within a complex less favorable for reduction than in the non-vesicle systems.     

Binding and fluorescence properties of the membrane domain of NADH-cytochrome-b5 reductase. Determination of the depth of Trp-16 in the bilayer

The membrane domain of NADH-cytochrome-b5 reductase, which extends from the amino-terminal myristic acid through the first 28 amino acid residues, can be isolated in cholate after a mild trypsin treatment of cholate-solubilized reductase, and in phospholipid vesicles after exhaustive trypsin treatment of vesicle-bound reductase. The detergent-solubilized peptide has a high affinity for phospholipid vesicles and can be reconstituted in vesicles by the detergent-dialysis method. The fluorescence of Trp-16 of this peptide is highly sensitive to the polarity of the microenvironment. The fluorescence quantum yield of this residue is 0.10 when the peptide is dispersed in 1% sodium cholate, but 0.46-0.52 when the peptide is reconstituted in phospholipid vesicles. Fluorescence energy transfer from this tryptophan residue in vesicle-bound peptide to a random array of acceptors in the head-group region of the vesicle outer monolayer shows that Trp-16 resides at a depth of 20-23 A in the bilayer.     

Pressure effects on the lateral distribution of cholesterol in lipid bilayers: a time-resolved spectroscopy study.

The effects of hydrostatic pressure and temperature on the phase behavior and physical properties of the binary mixture palmitoyloleoylphosphatidylcholine/cholesterol, over the 0-40 molar % range of cholesterol compositions, were determined from the changes in the fluorescence lifetime distribution and anisotropy decay parameters of the natural lipid trans-parinaric acid (t-PnA). Pressurized samples were excited with a Ti-sapphire subpicosecond laser, and fluorescence decays were analyzed by the quantified maximum entropy method. Above the transition temperature (T(T) = -5 degrees C), at atmospheric pressure, two liquid-crystalline phases, alpha and beta, are formed in this system. At each temperature and cholesterol concentration below the transition pressure, the fluorescence lifetime distribution pattern of t-PnA was clearly modulated by the pressure changes. Pressure increased the fraction of the liquid-ordered beta-phase and its order parameter, but it decreased the amount of cholesterol in this phase. Palmitoyloleoylphosphatidylcholine/cholesterol phase diagrams were also determined as a function of temperature and hydrostatic pressure.