Phospholipid composition and organization of cytochrome c oxidase preparations as determined by 31P-nuclear magnetic resonance

The molecular organization as well as the composition of the phospholipids in cytochrome c oxidase preparations (bovine heart) were investigated by 31P-nuclear magnetic resonance. In the so-called 'lipid-rich' preparation the lipids were found to form a fluid bilayer around the enzyme since the 31P-NMR spectrum was characteristic of a fast, axially symmetric motion of the phosphate groups with a chemical shift anisotropy of delta sigma = -45 ppm. In contrast, the 'lipid-depleted' cytochrome c oxidase gave rise to a broader spectrum where the motion of the phospholipids was no longer axially symmetric. Nevertheless, the total width of the spectrum was still considerably narrower than observed for immobilized phospholipids in solid crystals. Both enzyme preparations were dissolved in 1% detergent solution and used for high-resolution 31P-NMR spectroscopy. Narrow lines of about 20 Hz linewidth were obtained for both types of enzyme preparations, and well-resolved resonances could be assigned to cardiolipin, phosphatidylethanolamin and phosphatidylcholine. The major differences between lipid-rich and lipid-depleted cytochrome c oxidase were the absolute amount of phospholipid associated with the protein and the relative contribution of the individual lipid classes to the 31P-NMR spectrum. For lipid-rich cytochrome c oxidase about 130 molecules phospholipid were bound per enzyme (approx. 11 cardiolipins, 54 phosphatidylethanolamines and 64 phosphatidylcholines). For lipid-depleted cytochrome c oxidase only 6-18 lipids were bound per enzyme (1 or 2 cardiolipins, 3-8 phosphatidylethanolamines and 2-8 phosphatidylcholines). In contrast to earlier suggestions that cardiolipin is the only remaining lipid in lipid-depleted cytochrome c oxidase, the 31P-NMR studies demonstrate that all three lipids remain associated with the protein.     

Lipid bilayers in the gel phase become saturated by triton X-100 at lower surfactant concentrations than those in the fluid phase

It has been repeatedly observed that lipid bilayers in the gel phase are solubilized by lower concentrations of Triton X-100, at least within certain temperature ranges, or other nonionic detergents than bilayers in the fluid phase. In a previous study, we showed that detergent partition coefficients into the lipid bilayer were the same for the gel and the fluid phases. In this contribution, turbidity, calorimetry, and 31P-NMR concur in showing that bilayers in the gel state (at least down to 13-20°C below the gel-fluid transition temperature) become saturated with detergent at lower detergent concentrations than those in the fluid state, irrespective of temperature. The different saturation may explain the observed differences in solubilization.     

Characteristics of kinetics of phospholipid hydrolysis by phospholipase C from Bacillus cereus. Hydrolysis of phosphatidylcholine in the presence of deoxycholate

Using dynamic light scattering and 31P-NMR spectroscopy methods, the reaction of solubilization of phosphatidylcholine by the ionic detergent, sodium deoxycholate, in aqueous solutions was studied. The kinetics of phosphatidylchodine hydrolysis by phospholipase C from B. cereus depending on the size and structural organization of substrate aggregates was investigated. No phosphatidylcholine hydrolysis was observed in the case of lamellar organization of the substrate, the size of lamellas not exceeding 2000-5000 A. The substrate hydrolysis rate within mixed micelles was controlled by the accessibility of the substrate on the surface of micellar aggregates. There was a decrease in the phosphatidylcholine hydrolysis rate at high detergent concentrations in the system. It was concluded that such a decrease in the hydrolysis rate can be due to two reasons, i) the decrease in mixed micelle size with a simultaneous decrease of surface concentration of the substrate, and, ii) the formation of "pure" detergent micelles capable to adsorb the enzyme by decreasing the "effective" concentration of phospholipase C.     

NMR studies of a bacterial cell culture medium (LB Broth): cyclic nucleosides in yeast extracts

The composition of LB broth (tryptone, yeast extract and NaCl) was investigated by 1H,31P-NMR spectroscopy, FPLC and gel electrophoresis. An unexpected finding was the high level of 2'3'-cyclic nucleotides, detected by characteristic 31P-NMR resonances in the region 20-21 ppm, originating from the yeast component. 31P-NMR resonances for cyclic nucleotides were observed during the autolysis of Saccharomyces cerevisiae cells, and in model reactions of RNase with RNA.     

A 31P- and 2H-NMR study on lecithins in liquid crystalline polyoxyethylene detergents

Phosphatidylcholines were incorporated into hexagonal liquid cyrstalline mixtures of the non-ionic detergents Triton X-100 and octaethyleneglycoldodecylether with D₂O. It is shown by nuclear magnetic resonance (NMR) that the phospholipids adopt the hexagonal liquid crystalline structure of the detergent host lattice. The anisotropic motion of the phospholipid headgroups seems to be unaffected, whereas the acyl chains are disordered. Increasing phospholipid concentration leads to separation of a lamellar phase. The lamellar structure is also preferred at elevated temperatures. Phosphatidylcholines with saturated acyl chains undergo a transition from the hexagonal liquid crystalline to an ordered lamellar state. The shape of the ³¹P-NMR signals suggests that pure gel phase phospholipid separates out. The headgroup region of this gel phase phospholipid becomes immobilized after a few weeks of storage below the transition temperature as judged from ³¹P-NMR. At the same time ²H-NMR exhibits a new signal from D₆₂O undergoing slow isotropic motion. This behavior bears resemblance to the formation of a coagel in fatty acid-water systems.     

Induction of tumour hypoxia by a vasoactive agent. A combined NMR and radiobiological study

The effect of hydralazine treatment on 3 murine tumours (RIF-1, KHT and 16/C) was monitored using 31P-NMR. Changes in the 31P-NMR spectrum are compared with measurements of radiobiological hypoxic fraction (RHF) in the RIF-1 and KHT. Hydralazine is known to reduce temporarily blood flow in experimental tumours, and thus cause a transient increase in the RHF to 100% (in RIF-1 and KHT). This correlates with a decline in energy status as measured by 31P-NMR (i.e. there was an increase in Pi in all three tumours). Time-course data from the RIF-1 and KHT tumours show that maintenance of anaesthesia prolongs the hypoxia induced by hydralazine.     

Triton X-100 partitioning into sphingomyelin bilayers at subsolubilizing detergent concentrations: effect of lipid phase and a comparison with dipalmitoylphosphatidylcholine

We examined the partitioning of the nonionic detergent Triton X-100 at subsolubilizing concentrations into bilayers of either egg sphingomyelin (SM), palmitoyl SM, or dipalmitoylphosphatidylcholine. SM is known to require less detergent than phosphatidylcholine to achieve the same extent of solubilization, and for all three phospholipids solubilization is temperature dependent. In addition, the three lipids exhibit a gel-fluid phase transition in the 38-41 degrees C temperature range. Experiments have been performed at Triton X-100 concentrations well below the critical micellar concentration, so that only detergent monomers have to be considered. Lipid/detergent mol ratios were never <10:1, thus ensuring that the solubilization stage was never reached. Isothermal titration calorimetry, DSC, and infrared, fluorescence, and (31)P-NMR spectroscopies were applied in the 5-55 degrees C temperature range. The results show that, irrespective of the chemical nature of the lipid, DeltaG degrees of partitioning remained in the range of -27 kJ/mol lipid in the gel phase and of -30 kJ/mol lipid in the fluid phase. This small difference cannot account for the observed phase-dependent differences in solubilization. Such virtually constant DeltaG degrees occurred as a result of the compensation of enthalpic and entropic components, which varied with both temperature and lipid composition. Consequently, the observed different susceptibilities to solubilization cannot be attributed to differential binding but to further events in the solubilization process, e.g., bilayer saturability by detergent or propensity to form lipid-detergent mixed micelles. The data here shed light on the relatively unexplored early stages of membrane solubilization and open new ways to understand the phenomenon of membrane resistance toward detergent solubilization.     

Effects of the 'fusion peptide' from measles virus on the structure of N-methyl dioleoylphosphatidylethanolamine membranes and their fusion with Sendai virus.

31P nuclear magnetic resonance spectroscopy (31P-NMR) was used to study phospholipid organization in hydrated preparations of N-methyl dioleoylphosphatidylethanolamine and a 'fusion peptide' with the sequence: FAGV-VLAGAALGVAAAAQI, which corresponds to the amino terminus of the F1 subunit of the membrane fusion protein of measles virus. These amino acids are believed to mediate syncytia formation, host-cell penetration and hemolysis by infectious virus. The presence of the peptide at 0.5 mole percent significantly facilitated the formation of isotropic 31P resonances. The effects at 1 mole percent peptide were substantially enhanced over the effects observed at 0.5 mole percent, leading to a decrease in the onset temperature of the formation of the isotropic 31P-NMR resonances by about 30 degrees C. The formation of such isotropic 31P-NMR resonances has been previously associated with an increased rate of fusion of large unilamellar vesicles composed of N-methyl dioleoylphosphatidylethanolamine. Enhanced fusion of octadecyl rhodamine-labelled Sendai virus with N-methyl dioleoylphosphatidylethanolamine large unilamellar vesicles was observed when the 'fusion peptide' was incorporated into the large unilamellar vesicles.     

The interaction of phosphatidylcholine bilayers with Triton X-100

The interaction of multilamellar phosphatidylcholine vesicles with the non-ionic detergent Triton X-100 has been studied under equilibrium conditions, specially in the sub-lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X-100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X-100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration. The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier-transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H-NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier-transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel-to-fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P-NMR and 2H-NMR spectra, and a new low-temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X-100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one-component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.     

Light-induced membrane protein phosphorylation in the bovine rod outer segment. A magic angle spinning 31P-NMR study

Magic angle spinning 31P-NMR (MAS 31P-NMR) spectra of bovine rod outer segments, unphosphorylated and phosphorylated, were obtained. In the phosphorylated samples the spectra showed new resonances not assignable to phospholipids. These signals were present only when stimulation of receptor phosphorylation occurred. These resonances were not due to exogenous, soluble phosphorus-containing compounds. Limited proteolysis to remove the carboxyl-terminal region of the photoreceptor that contains the phosphorylation sites removed these resonances. The chemical shifts were in the usual range for serine phosphate and threonine phosphate. The pKa obtained from a pH titration of the 31P chemical shift was typical of serine phosphate. Therefore, these 31P-NMR resonances were assigned to the phosphorylation sites on membrane proteins in the rod outer segment disk membranes. Static 31P-NMR measurements revealed that at least some of these sites gave rise to relatively narrow resonances, indicative of considerable motional freedom of the carboxyl-terminal segment of the photoreceptor when phosphorylated. These data indicate that it is possible to study phosphorylation sites on membrane proteins using MAS 31P-NMR, and that using in vivo 31P 'spin labelling' one can study directly and selectively regions of receptors crucial to receptor function.     

Parameters influencing the determination of liposome lamellarity by 31P-NMR

The lamellarity of liposomes influences to a great extent the encapsulation efficiency, the efflux rate of liposomally encapsulated material, and the fate of a drug after cellular uptake. 31P-NMR in combination with the use of chemical shift reagents has been described for the determination of lamellarity of liposomes and this study was performed to evaluate the applicability of 31P-NMR analysis as published in the past. To date, very few details about the required conditions throughout the measurements are known. In this study the influence of various parameters on the measurement, such as different buffers with changing ion concentrations, varying pH and different shift reagents at increasing concentrations was investigated. Results were discussed by using cryo-electronmicroscopy as a reference method. The data of this study show that 31P-NMR might not result in the correct determination of liposome lamellarity, depending on the experimental settings and the shape of the liposomes.     

NMR-invisible ATP in rat heart and its change in ischemia

The subcellular compartmentalization of adenosine 5'-triphosphate (ATP) in isolated perfused rat heart and its relation to energy depletion in ischemia were examined by 31P nuclear magnetic resonance (31P-NMR) spectroscopy and chemical analyses. The signal intensities of the beta-phosphate of ATP and creatine phosphate in the 31P-NMR were standardized by the intracellular volume ratio measured with 23Na-NMR to determine the actual content of each. During aerobic perfusion the ATP content determined by NMR (13.7 +/- 2.2 mumol/g dry weight) was significantly lower than that found by chemical analysis (22.4 +/- 0.7 mumol/g dry weight), while the creatine phosphate contents determined by the two methods were the same. During ischemia at 33 degrees C, the signal of the beta-phosphate of ATP in the 31P-NMR spectrum decreased progressively, disappearing completely after 16 min. But at this time 5.7 +/- 1.7 mumol/g dry weight of myocardial ATP was still detected by chemical analysis. These results indicated that there were two different compartments of intracellular ATP in the heart, only one of which is detectable by 31P-NMR spectroscopy, and that during ischemia the ATP that is detectable, which seems to be the free ATP in the cytosol, decreased more rapidly than the ATP in the other compartment.     

NMR studies of the phosphoserine regions of bovine alpha s1- and beta-casein. Assignment of 31P resonances to specific phosphoserines and cation binding studied by measurement of enhancement of 1H relaxation rate

(1) High-resolution 31P-NMR was used to study the environment of the phosphoserine residues of the phosphoproteins, alpha s1-casein B, beta-casein A2 and beta-casein C. For reference purposes 31P-NMR spectra of phosvitin and ovalbumin were also collected. (2) 31P resonances were assigned to specific phosphoserine residues as a result of comparisons of the high-resolution 31P-NMR spectra for alpha s1- and beta-caseins and for peptide fragments of these proteins obtained by cyanogen bromide and trypsin cleavage. (3) Measurements of the enhancement of the relaxation rate for water protons (1H) on addition of Mn2+ to alpha s1-casein B and to a fragment alpha s1-CN3, obtained by cyanogen bromide cleavage, gave approximate pK values for the binding groups and suggest the possibility of a conformational change induced by varying the concentration of divalent cation.     

Mevalonate-Dependent Enzymatic Synthesis of Amorphadiene Driven by an ATP-Regeneration System Using Polyphosphate Kinase

Polyphosphate kinase (PPK), which can regenerate ATP from ADP, was utilized in the mevalonate-dependent enzymatic synthesis of amorphadiene. The activity of PPK, cloned from Escherichia coli, was determined by (31)P-NMR. The yield from the PPK-catalyzed synthesis was 25%, 2.5 times higher than that without PPK. The (31)P-NMR analysis of the final reaction mixture indicated no accumulation of intermediates.     

The binding of physiologically significant protons to 2,3-diphosphoglycerate

The intrinsic pKa values of protons of 2,3-diphosphoglycerate (DPG) which titrate in the physiologically significant range (i.e., pH 6.8-7.8) have been determined by measuring the changes in chemical shifts of the two phosphate resonances of the molecule as a function of pH using 31P-NMR spectroscopy. While conventional acid-base titration techniques resulted in apparent pKa values of 6.39 and 7.39 for these protons, analysis of the 31P-NMR data by statistical thermodynamic methods yielded intrinsic pKa values of 6.99 +/- 0.07 and 7.28 +/- 0.04, for protons associated with the phosphates bound to carbon-3 (C-3) and carbon-2 (C-2), respectively, with an interaction energy of +0.77 kcal/mol. The free energies for the binding of protons to the C-2 and C-3 phosphates and the associated interaction energies determined by 31P-NMR were used to generate a theoretical titration curve which was essentially identical to that determined by conventional acid-base titration. The physiological implications of this work are briefly discussed.     

Phosphorus nuclear magnetic resonance of Acholeplasma laidlawii cell membranes and derived liposomes.

1. The 129 MHz 31P-NMR spectrum of Acholeplasma laidlawii membranes is very similar to the spectrum of the derived liposomes and is a typical "solid state" spectrum in which the major contribution to the linewidth is made by the chemical shift anisotropy. From the value of the chemical shift anisotropy an order parameter of 0.15 is estimated for the lipid phosphates in both membranes. 2. The 31P-NMR spectrum of the A. laidlawii membrane is insensitive to pronase digestion of 4-60% of the membrane proteins and subsequent cytochrome C binding. These results indicate that either no strong lipid polar headgroup-protein interactions occur in the membrane or that the lipid-protein "complexes" in the membrane have a fast rotation (Tc shorter than 10(-6)S) along an axis perpendicular to the plane of the membrane. 3. Phospholipase A2 degrades all the phosphatidylglycerol in the membrane. The resulting membrane contains a phosphoglycolipid as the sole phosphorus-containing compound. The 31P-NMR spectrum of these membranes is identical to the spectrum of the native membranes suggesting a similar motion for the phosphate groups in both lipids. 4. Ca2+ binding to liposomes prepared from either the total polar lipids or the total phosphorus-containing lipids isolated from the A. laidlawii membrane does not affect the 21P-NMR spectrum. 5. The 31P-NMR spectrum of the membranes and derived liposomes, however, is sensitive to lipid phase transitions. When the membrane lipids are in the gel state a broadening of the 31P resonance occurs demonstrating that the polar head group motion in a biological membrane is more restricted below the lipid-phase transition temperature.     

H~+诱导心磷脂的六角形Ⅱ相和H~+的跨膜转运

本文报告H~ 能诱导心磷脂由双层排列转变为六角形Ⅱ相.含心磷脂的多层脂囊泡的~(31)P中核磁共振谱显示高场峰低场肩的双层排列特点,当pH降到2时,~(31)P核磁共振谱表现为低场峰高场肩的六角形Ⅱ相特点,表明H~ 对心磷脂多形性转变的诱导作用.用oxonol-V作为探剂.H~ 可使结合在人工脂膜上的oxonl-V的吸收峰红移和光吸收增加,表明心磷脂的六角形Ⅱ相在人工脂膜上具有H~ 的载体特性,易化H~ 的跨膜转运.     

Localization and identification of the compound causing peak ‘X’ in the 31P-NMR spectrum of Saccharomyces cerevisiae

The present study is aimed at identifying the unidentified compound which gives rise to the so-called resonance ‘X’ in the ³¹P-NMR spectra of yeast cells. In addition, it is attempted to determine the localization of X (inside or outside the cell). Enzymic removal of the cell wall causes resonance ‘X’ to disappear in the spectra of the cells. This observation indicates an extracellular localization of X. The ³¹P-NMR spectrum of the phosphomannan extracted from the yeast shows a single resonance at exactly the same position as that of resonance ‘X’. Extraction of the phosphomannan from delipidized cells causes resonance ‘X’ to disappear from the ³¹P-NMR spectrum of the cells. The intensity of resonance ‘X’ in the spectrum of the intact cells can be almost quantitatively attributed to the amount of phosphomannan present in the yeast. The present results demonstrate that the resonance ‘X’ in the ³¹P-NMR spectrum of yeast cells is caused by phosphomannan in the cell wall.     

The 31P-NMR spectrum of the dodecamer d(GACGATATCGTC).

The resonances in the 31P-NMR spectrum of the dodecamer d(GACGATATCGTC) have been assigned by regiospecific labelling with oxygen-17. All 11 resonances are clearly resolved at 26 degrees C. Most noticeably, individual resonances of the dinucleoside phosphates d(CpG), d(TpC), d(GpA) and d(ApT) which occur more than once can clearly be distinguished. This indicates that the position of the phosphate group in the oligomer influences its 31P-NMR shift. This observation is in agreement with what has been found for the 31P-NMR spectra of d(CGCGAATTCGCG) [Ott, J. and Eckstein, F. (1985) Biochemistry 24] and d(GGAATTCC) [Connolly, B.A. and Eckstein, F. (1984) Biochemistry 23, 5523-5527]. In general, the chemical shift appears the more at higher field the more central the dinucleoside phosphate is located in the oligomer. Exceptions are the resonances of dinucleoside phosphates of the type 5'-PyPu-3' which appear at lower field than expected from this rule. A reasonable correlation between 31P-NMR chemical shifts and the sum function of the base plane roll angles derived from Calladine's rule [Calladine, C.R. (1982) J. Mol. Biol. 161, 343-352] exists.     

Effects of the anti-cancer drug adriamycin on the energy metabolism of rat heart as measured by in vivo 31P-NMR and implications for adriamycin-induced cardiotoxicity

In vivo 31P-NMR was used to measure the effects of the anti-tumor drug adriamycin on the energy metabolism of rat heart. The exclusive acquisition of NMR signal from cardiac muscle was assured by positioning a solenoidal radio-frequency NMR coil around the heart. Appropriate control experiments verified that 31P-NMR spectra solely originated from this organ. Acute effects occurring shortly after adriamycin administration are expressed in 31P spectra as a dose-dependent decline in the cardiac levels of phosphocreatine, after which stabilization at a new steady-state level occurs. These acute effects of a single dose are complete in 30-60 min and no significant further changes take place within 150 min after drug introduction. Longer-term effects of single high doses and of multiple lower doses were measured up to a week after the initiation of treatment. It seemed that at a total dose of 20 mg/kg, drug-induced interference with cardiac energy metabolism was more pronounced than at the same dose in the acute phase. These 31P-NMR data demonstrate that adriamycin treatment is accompanied by a decrease of the cardiac phosphocreatine/ATP ratio which might be an expression of the well-established cardiotoxicity of the drug.