Phase behavior of the major lipids of Tetrahymena ciliary membranes

The major lipids of Tetrahymena membranes have been purified by thin-layer and high pressure liquid chromatography and the phosphatidylethanolamine and aminoethylphosphonate lipids were examined in detail. ³¹P-NMR, X-ray diffraction and freeze-fracture electron microscopy were employed to describe the phase behavior of these lipids. The phosphatidylethanolamine was found to form a hexagonal phase above 10°C. The aminoethylphosphonate formed a lamellar phase up to 20°C, but converted to a hexagonal phase structure at 40°C. Small amounts of phosphatidylcholine stabilized the lamellar phase for the aminoethylphosphonate. ³¹P-NMR spectra of the intact ciliary membranes were consistent with a phospholipid bilayer at 30°C, suggesting that phosphatidylcholine in the membrane stabilized the lamellar form, even though most of the lipid of that membrane prefers a hexagonal phase in pure form at 30°C. ³¹P-NMR spectra also showed a distinctive difference in the chemical shift tensor of the aminoethylphosphonolipid, when compared to that of phosphatidylethanolamine, due to the difference in chemical structure of the polar headgroups of the two lipids.     

Effects of adriamycin on lipid polymorphism in cardiolipin-containing model and mitochondrial membranes

(1) The effects of the anti-tumor drug adriamycin on lipid polymorphism in cardiolipin-containing model membranes and in isolated inner mitochondrial membranes has been examined by ³¹P-NMR. (2) Adriamycin binding does not affect the macroscopic structure or local order in the phosphate region of cardiolipin liposomes. (3) In cardiolipin liposomes and in cardiolipin-phosphatidylcholine (1:1) liposomes, the drug inhibits the ability of Ca²⁺ to induce the hexagonal H_II phase. (4) Adriamycin interaction with both dioleoylphosphatidylethanolamine-cardiolipin (2:1) and dioleoylphosphatidylethanolamine-phosphatidylserine (1:1) liposomes results in structural phase separation into a liquid-crystalline hexagonal H_II phase for the phosphatidylethanolamine and a liquid-crystalline lamellar phase for the negatively charged phospholipid. (5) Combined high-resolution ³¹P-NMR, electron microscopy and light scattering studies reveal the prominent fusion capacity of adriamycin towards cardiolipin-phosphatidylcholine small unilamellar vesicles. (6) Addition of Ca²⁺ to total rat liver inner mitochondrial membrane lipids, dispersed in excess buffer, results in hexagonal H_II formation for part of the phospholipids. By contrast, the original bilayer structure is completely conserved when the above experiment is performed in the presence of adriamycin. (7) ³¹P-NMR spectra of isolated inner mitochondrial membranes are indicative of a bilayer organization for the majority of the phospholipids. Approximately 15% of the signal intensity originates from phospholipids which experience isotropic motion. Adriamycin addition almost completely eliminates the latter spectral component. In the absence of adriamycin, Ca²⁺ addition greatly increases the percentage of the phospholipids giving rise to an isotropic signal possibly indicating the formation of non-lamellar lipid structures. Adriamycin which specifically binds to cardiolipin (K. Nicolay et al. (1984) Biochim. Biophys. Acta 778, 359–371) completely blocks the Ca²⁺-induced structural reorganization of the lipids in this membrane.     

Phase behavior of isolated photoreceptor membrane lipids is modulated by bivalent cations

The phase behavior of isolated photoreceptor membrane lipids is further investigated by ³¹P-NMR, in view of earlier discrepant results [(1979) Biochim. Biophys. Acta 558, 330–337; (1982) FEBS Lett. 124, 93–99]. We present evidence that the discrepancy is due to bivalent cations. When resuspended in aqueous media at neutral pH in the absence of bivalent cations, the isolated photoreceptor membrane lipids largely adopt the bilayer configuration. However, upon addition of such cations (Ca²⁺ Mg²⁺) or when resuspended in their presence, the formation of other phases (hexagonal H_II, lipidic particles) results. The rate of this transition depends on cation concentration and temperature. The transition is not easily reversed by addition of EDTA. Implications with regard to photoreceptor membrane structure and function need further study.     

Effects of divalent cations and pH on phosphatidylserine model membranes: A 31P NMR study

The influence of divalent cations, and pH on the behaviour of phosphatidylserine, derived from egg phosphatidylcholine, has been examined employing ³¹P-NMR techniques. The addition of Ca²⁺ results in the observation of a “rigid lattice” ³¹P-NMR spectra and more than an order of magnitude increase in the spin-lattice relaxation time T₁. This corresponds to a strong and specific headgroup immobilization by Ca²⁺, similar to that observed for anhydrous phosphatidylserine. At pH 7.4 the hydrated sodium salt of (egg) phosphatidylserine adopts the bilayer phase, whereas when the pH is decreased through 3.5 a bilayer to hexagonal (H_II) polymorphic phase transition is observed at 50°C, which is unaffected by equimolar cholesterol. The same transition is shown to occur at 37°C for phosphatidylserine isolated from human erythrocytes.     

Fast reversal of the initial reaction steps of the plasma membrane (Ca2+ + Mg2+)-ATPase

(1) Calmodulin-depleted red cell membranes catalyse a Ca²⁺, Mg²⁺-dependent ATP-[³H]ADP exchange at 37° C. The Ca²⁺, Mg²⁺-dependent exchange, measured at 20 μM CaCl₂, 1.5 mM MgCl₂, 1.5 mM ADP and 1.5 mM ATP, is comparable to the (Ca²⁺ + Mg²⁺)-ATPase activity, between 0.3 and 0.8 mmol/litre original cells per h. (2) EDTA-washed membranes present a Ca²⁺-dependent ATP-ADP exchange whose rate is not more than 7% of that found in a Mg²⁺-containing medium, while their Ca²⁺-dependent ATPase is essentially zero. Addition of 1.5 mM MgCl₂ to the medium restores both activities to the levels found with membranes not treated with EDTA. (3) Calmodulin (16 μg/ml) produces an eight-fold stimulation of the Ca²⁺-dependent ATP-ADP exchange, slightly less than it stimulates the Ca²⁺-dependent ATP hydrolysis. The effect of 1.5 mM MgCl₂ on the exchange is greater in the presence than in the absence of calmodulin. (4) It is proposed that the reversal of the initial phosphorylation of the Ca²⁺ pump, occurring at a fast rate at 37° C, involves a conformational change in the phosphoenzyme. Thus, it would be an ADP-liganded phosphoenzyme of the form EP(ADP) that would experience the fast conformational transition at 37° C. The great difficulty in producing an overall reversal of the Ca²⁺ pump should then be due to one or more reaction steps later than and including Ca²⁺ release and dephosphorylation.     

31P-NMR studies on phospholipid structure in membranes of intact,functionally-active,rat liver mitochondria

³¹P-NMR studies of intact functional rat liver mitochondria at 37°C demonstrate that the large majority (?95%) of endogenous phospholipids exhibit motional properties consistent with bilayer structure. This property is unaffected by oxidative phosphorylation processes or the presence of Ca²⁺.     

The sea urchin extraembryonic hyaline layer: Ionic parameters controlling the hyalin gelation reaction

• 1.1. As reported previously (Robinson, 1988) the Ca²⁺-induced self-association reaction of the protein hyalin, purified from the sea urchin extraembryonic hyaline layer, was modulated by both Mg²⁺ and NaCl.
• 2.2. In the presence of 400 mM NaCl the apparent dissociation constant (Ca²⁺) decreased five-fold from 4.8 ± 1.1 mM in the absence to 0.9 ± 0.5 mM in the presence of 20 mM Mg²⁺.
• 3.3. The potentiating effect of Mg²⁺ occurred with an apparent dissociation constant (Mg²⁺) of 4.6 ± 0.5mM.
• 4.4. In the absence of Ca²⁺ or NaCl hyalin dissociated from isolated hyaline layers indicating that the behavior of hyalin within the layer is predictable from results obtained with the purified protein.

     

Regulation of the shape of unsealed erythrocyte membranes by Mg-ATP and Ca2+

In the presence of MgCl₂ and ATP, the specific viscosity of suspensions of unsealed freezethawed erythrocyte membranes decreased slowly with time at 37 °C. The decrease in viscosity was found to be an index of Mg-ATP-specific induced folding of these membranes. Mg-ATP-dependent shape or viscosity changes were found to be highly temperature dependent and the viscosity of these membranes did not decrease in the presence of 2 mm 5′-adenyl imidodiphosphate and MgCl₂. Cyclic AMP, NaCl, or KCl did not have any effect on the rate of Mg-ATP-induced viscosity decreases. The Mg-ATP-dependent viscosity decreases were inhibited 100% by 1 mm chlorpromazine or 1 mmN-ethylmaleimide. Mg-ATP-dependent viscosity decreases were half-maximally inhibited by 1 μm Ca²⁺ and completely inhibited by 3–5 μm Ca²⁺. Ca²⁺ (5 μm) also inhibited Mg²⁺-dependent phosphorylation 25 to 30% in these membranes. However, if these membranes were preincubated in the absence of Ca²⁺ for greater than 10 min at 37 °C, 5 μm Ca²⁺ no longer inhibited Mg-ATP-dependent viscosity decreases and only inhibited Mg²⁺-dependent phosphorylation 5% in these preincubated membranes. Preincubation of these membranes at 37 °C for 10 min in the absence of Ca²⁺ also resulted in the loss of approximately 40 to 50% of the high-Ca²⁺ affinity Ca + Mg-ATPase activity. The presence of 5 μm Ca²⁺ in the preincubation medium protected against the loss of the inhibitory effect of Ca²⁺ on Mg²⁺-dependent phosphorylation and Mg-ATP-dependent viscosity decreases. The presence of Ca²⁺ in the preincubation medium also protected against the loss of Ca + Mg-ATPase activity in these membranes. It is hypothesized that freeze-thawed erythrocyte membranes contain a Ca²⁺ phosphatase activity which is temperature labile in the absence of Ca²⁺ and that this Ca²⁺ phosphatase activity may be involved in the regulation of shape of these membranes. Also discussed is the possible relationship of this Ca²⁺ phosphatase with Ca + Mg-ATPase activity and the problems inherent in studying Ca²⁺-regulated functions in freeze-thawed erythrocyte membranes.     

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by P-NMR

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing.In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for ³¹P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 °C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 °C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.     

Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A P-NMR study

1. 1. The ³¹P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.

2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (H_II) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the H_II) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.

3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit ³¹P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca²⁺ results in hexagonal (H_II) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.

4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit ³¹P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca²⁺ results in an increase in the size of this component, which can become the dominant spectral feature.

5. 5. Intact mitochondria, at 4°C, exhibit ³¹P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, P_i, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.

6. 6. It is suggested that the uncoupling action of high Ca²⁺ concentrations on intact mitochondria may be related to a Ca²⁺-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

Phosphorylation of skeletal muscle myosin light chain kinase by the catalytic subunit of cAMP-dependent protein kinase

Phosphatidylethanolamine (PE) was isolated from membranes of Bacillus megaterium. The organism was grown at 20°C and 55°C. The phase equilibria in PE/water systems were studied by ²H and ³¹P nuclear magnetic resonance, and by polarized light microscopy. PE isolated from B. megaterium grown at 20°C forms a lamellar liquid crystalline phase at the growth temperature, and at low water contents a cubic liquid crystalline phase at 58°C. The ratio iso/ante-iso acyl chains was 0.3 in this lipid. PE isolated from this organism grown at 55°C forms only a lamellar liquid crystalline phase up to at least 65°C. In this lipid the ratio iso/ante-iso acyl chains was 3.2.     

Interactions of La3+ with phosphatidylserine vesicles Binding,phase transition,leakage, 31P-NMR and fusion

The interaction of La³⁺ with phosphatidylserine vesicles is studied by differential scanning calorimetry, ¹⁴⁰La binding, ³¹P-NMR chemical shifts and relaxation rates, carboxyfluorescein and [¹⁴C]sucrose release, X-ray diffraction and freeze-fracture electron microscopy. In the presence of La³⁺ concentrations above 1 mM and an incubation temperature of 38°C, i.e., at the phase transition temperature of the complex La/phosphatidylserine, the binding ratio of La/lipid exceeds a $\text{1}\text{3}$ ratio, reaching saturation at a $\text{1}\text{2}$ ratio. Analysis, employing a modified Gouy-Chapman equation, indicates a significant increase in the intrinsic binding constant of La/phosphatidylserine when the La³⁺ concentration exceeds the threshold concentration for leakage. The analysis illustrates that at the molecular level the binding of La³⁺ can be comparable to or even weaker than that of Ca²⁺, but that even when present at smaller concentrations La³⁺ competes with and partially displaces Ca²⁺ from membranes or other negatively charged surfaces. The results suggest that the sequence La³⁺>Ca²⁺>Mg²⁺ reflects both the binding strength of these cations to phosphatidylserine as well as their ability to induce leakage, enhancement of ³¹P spin-lattice relaxation rates, fusion and other structural changes. The leakage, fusion, and other structural changes are more pronounced at the phase transition temperature of the La/lipid complex.     

Ca2+-induced phase separation in phosphatidylserine,phosphatidylethanolamine and phosphatidylcholine mixed membranes

Ca²⁺-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidyl-ethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94–100% at 23°C and 84–88% at 40°C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74–85% at 23°C and 61–79% at 40°C. Ca²⁺ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was $\text{1.4 · 10}{}^{\mathrm{?7}}$ M for phosphatidylserine-phosphatidylethanolamine and $\text{1.2 · 10}{}^{\mathrm{?6}}$ M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca²⁺ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17°C) and phosphatidylcholine (?15°C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca²⁺-chelated solid-phase phosphatidylserine domain.     

A fluorescence polarization study of calcium and phase behaviour in synaptosomal lipids

Steady-state fluorescence polarization of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene reported temperature-dependent lipid order in l-α-dimyristoylphosphatidylcholine, egg phosphatidylcholine and synaptosomal membranes. No change in lipid order was detected after depolarization of synaptosomes by veratridine (150 μM) even in the presence of 2 mM CaCl₂. However, Ca²⁺ reduced the mobility of a second probe, dansylated dipalmitoylphosphatidylethanolamine, in dispersions of synaptosomal lipids. This effect, which was seen at a Ca²⁺/total phospholipid ratio as low as 0.1, may represent an interaction between the cation and negatively-charged phospholipids. It is suggested that Ca²⁺ promotes a phase separation in synaptosomal lipids which may be relevant to the process of neurotransmitter release.     

31P-NMR observation of the temperature and glycerol induced non-lamellar phase formation in wheat thylakoid membranes

³¹P-NMR spectra at 162 MHz were used to monitor phase changes of wheat thylakoid membranes as a function of temperature. At room temperature the³¹P-NMR line was a superposition of anisotropic component characteristic of phospholipid lamellar phase and isotropic line due to inorganic phosphorus or small membrane vesicles arising as an effect of preparation. For temperatures higher than +35 °C an increase of the isotropic component occurs, which is irreversible as the sample is cooled. For the temperatures between +55 °C and +60 °C the presence of the hexagonal phase cylinders is suggested, as monitored by phosphorus lineshape. However, the addition of glycerol stimulates a formation of the isotropic phase. The effect of reconstitution of freeze-dried thylakoid membranes by addition of water or water-glycerol medium to the sample was examined. As lyophilizate was gradually diluted, the increase of isotropic line component was observed. For thylakoid membranes suspended in D₂O at the highest dilution examined, the line contribution due to small membrane fragments is not greater than 50%, but in presence of glycerol, this contribution could reach 70%. This suggests that the presence of glycerol increases the formation of the small membrane particles as the thylakoid membrane is reconstituted from lyophilizate. The wheat thylakoid membranes reconstituted from lyophilizate show, in comparison to native membranes, the increased contribution of small membrane vesicles. Moreover, the³¹P -NMR spectra suggest the appearance of the hexagonal phase cylinders even at +50 °C.Abbreviations DGDG digalactosyldiacylglycerol - DLPC dilinoleoyl phosphatidylcholine - DLPE dilinoleoyl phosphatidylethanolamine - EDTA ethylenediamine-tetraacetic acid - MGDG monogalactosyldiacylglycerol - NMR nuclear magnetic resonance - PC phosphatidylcholine - PG phosphatidylglycerol - PSII photosystem II - TGDG trigalactosyldiacylglycerol - Tris Tris-(hydroxymethyl)-aminomethan - S/N signal to noise ratio     

Ca2+ transport in mitochondria of the ciliate protozoan Tetrahymena pyriformis

Mitochondria isolated from the late-exponential non-shaken culture of the ciliate protozoan Tetrahymena pyriformis GL was investigated. The presence of energy-dependent Ca²⁺ transport system was shown. In the main the properties of this system have been essentially the same as in mitochondria of vertebrate organisms. The isolated mitochondria contained 23±5 ng-ion Ca²⁺ per mg of protein. The intramitochondrial free concentration of Ca²⁺ was measured in the presence of uncoupler FCCP with the use of fluorescent Ca²⁺ chelator chlortetracycline and null point titration method. In the absence of phosphate, free [Ca²⁺] varied from 1 to 2.5 mM depending on the internal Ca²⁺ content. In the presence of 2 mM phosphate, free [Ca²⁺]_in has not exceeded 0.1–0.3 mM. It was shown that ruthenium red and Mg²⁺ in different manner have an inhibitory effect on Ca²⁺ transport. Besides this, Mg²⁺ also has a stabilizing effect on mitochondria, possibly, by preventing passive ions leaks across the membrane.