Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase

Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of alkyl chain length and introduction of unsaturation in the alkyl chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine- and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82 degrees C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and III reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and III. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit magnetic parameters of cholestane spin labels in membranes from Q-band second-derivative ESR spectra to monitor the dielectric environment around the nitroxide radical, it is concluded that cholesterol incorporation increases water accessibility in the hydrophilic loci of the membrane. In contrast, 12-(9-anthroyloxy)stearic acid fluorescence showed that water accessibility is decreased in the hydrophobic loci of the membrane.     

Racemization in the Coupling Reaction with the Several Methods beside the Activated Ester Methods

The degree of racemization in the coupling reaction, Pro-Val + Pro, with the several other methods than the activated ester methods was measured and the results were compared with that in the coupling reaction, Leu-Phe + Val, as well as in the previous paper.¹⁾ In the azide and the mixed anhydride methods, no or almost no racemization was detected, whereas in the other tested methods of peptide synthesis (Pachornik-, DCCD- and phosphazo-methods) the significantly large racemization was observed. It can be attributed to the strong nucleo- philic N-atom in the penultimate amino acids (Pro) and the steric hindrance of C-terminal amino acid (Val), which are favourable to the formation of the oxazolone ring.

This assumption was further systematically confirmed in the synthesis of the other several tripeptides with the DCCD method. The separation of the diastereoisomers (LLL and LDL) of the resulting tripeptides by gas chromatography with a packed column was also here reported.     

Dynamic fluorescence quenching studies on lipid mobilities in phosphatidylcholine-cholesterol membranes

Bimolecular collision rate of 8-anilinonaphthalene-1-sulfonic acid (ANS) and the nitroxide doxyl group attached to various carbons on stearic acid spin labels (n-SASL) in phosphatidylcholine-cholesterol membranes in the fluid phase was studied by observing dynamic quenching of ANS fluorescence by n-SASL's. The excited-state lifetime of ANS and its reduction by the n-SASL doxyl group were directly measured by the time-correlated single photon counting technique to observe only dynamic quenching separately from static quenching and were analyzed by using Stern-Volmer relations. The collision rate of ANS with the n-SASL doxyl group ranges between 1 X 10(7) and 6 X 10(7), and the extent of dynamic quenching by n-SASL is in the order of 5-much much greater than 6- greater than 7- less than 9- less than 10- less than 12- less than 16-SASL (less than 5-SASL) in dimyristoylphosphatidylcholine (DMPC) membranes. Collision rate of 16-SASL is only 10% less than that of 5-SASL. Since the naphthalene ring of ANS is located in the near-surface region of the membrane, these results indicate that the methyl terminal of SASL appears in the near surface area frequently, probably due to extensive gauche-trans isomerism of the methylene chain. The presence of 30 mol% cholesterol decreases the collision rate of ANS with 12- and 16-SASL doxyl groups but not with the 5-SASL doxyl group in DMPC membranes. On the other hand, in egg-yolk phosphatidylcholine membranes, inclusion of 30 mol% cholesterol does not affect the collision of ANS with either 5-SASL or 16-SASL doxyl groups, in agreement with our previous observation that alkyl chain unsaturation moderates cholesterol effects on lipid motion in the membrane (Kusumi et al., Biochim. Biophys. Acta 854, 307-317). It is suggested that dynamic quenching of ANS fluorescence by lipid-type spin labels is a useful new monitor of membrane fluidity that reports on various lipid mobilities in the membrane; a class of motion can be preferentially observed over others by selecting a proper spin label, i.e., rotational diffusion of lipid about its long axis and translational diffusion by using 5-SASL, wobbling motion of the lipid long axis by using 7-SASL or androstane spin label, and gauche-trans isomerism by using 16-SASL.     

Activation of mouse peritoneal macrophages by synthetic glyceroglycolipid liposomes

Summary Liposomes composed of chemically synthesized glyceroglycolipids, such as 1,2-dipalmityl-[-cellobiosyl-(1 3)]-glycerol (Cel-DAG), 1,2-dipalmityl-[-lactosyl-(1 3)]-glycerol, or 1,2-dipalmityl-[-maltosyl-(1 3)]-glycerol, were found to enhance protective immunity against transplantable tumor cells (sarcoma 180) in ICR mice. Peritoneal exudate cells prepared from mice treated in vivo with Cel-DAG showed cytostatic activity in vitro against the mouse leukemia cell line, EL-4. Adherent cells separated from this preparation showed similar activity. Peritoneal cells from polypeptone-injected mice acquired appreciable cytostatic activity when incubated in vitro in the presence of glyceroglycolipid liposomes. The adherent cell fraction alone showed rather weak cytostatic activity when pretreated with the glyceroglycolipids, and full activity was restored by supplementing with the nonadherent cell fraction. The ability of glycolipids to induce tumoricidal effects was affected by cholesterol content: with increasing cholesterol content, the activities decreased. Cholesterol-free glycolipid liposomes were taken more efficiently by macrophages than cholesterol-containing liposomes. Cholersterol modifies the surface property of glyceroglycolipid liposomes. Activation of macrophages is responsible for enhancement of protective immunity against tumor cells by injection of these glycolipids in vivo.This work was supported in part by Grants-in-Aid (Nos. 58010010, and 59870076) for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Mapping and disruption of the cybB gene coding for cytochrome b561 in Escherichia coli

Abstract The cybB gene on a plasmid encoding cytochrome b ₅₆₁ in Escherichia coli was disrupted by insertion of Km^rl determinant DNA. The cromosomal cybB gene was replaced by the inactivated cybB gene on the plasmid by homologous recombination using λ phage lysogenization and heat-induction. The replacement was confirmed by Southern and Western blotting analyses. Deficiency on the cybB gene product did not affect the growth properties of the cells, and the oxidase activities of the cells dependent on various substrates were similar to those of the parental strain. Cytochrome b ₅₆₁ is concluded to be expressed in E. coli , but may not play a major role in cell growth. In the genetic map of E. coli , the cybB gene was determined by conjugational and transductional crosses to be at 31 min between trg and terC .     

Exposure of Leaves of Cucumis sativus L. to Low Temperatures in the Light Causes Uncoupling of Thylakoids II. Non-Destructive Measurements with Intact Leaves

To examine the effects of chilling of leaves of cucumber (Cucumissativus L.) in moderate light on the coupling state of thylakoidsin situ, changes in fluorescence, changes in light scatteringand flash-induced changes in absorbance at 518 nm were examinedin intact leaves. After chilling of leaves at 5?C in the lightfor 5 h, the non-photochemical quenching of fluorescence, ameasure of energisation of thylakoids, was largely suppressed.The treatment also caused a suppression of light-induced changesin the light scattering by leaves, which depends on the formationof a pH gradient across thylakoid membranes. When thylakoidswere prepared by very gentle methods from the leaves chilledin the light, through a step of preparation of intact chloro-plasts,the transport of electrons from H₂O to ferricyanide was uncoupled,being insensitive to an uncoupler, methylamine. These data provide consistent evidence that the thylakoids areuncoupled in situ by the chilling of leaves in the light and,as a consequence of the uncoupling, the energisation of themembranes is suppressed. However, the decay of the flash-inducedchange in absorbance at 518 nm in leaves was not markedly acceleratedby the treatment. The thylakoids isolated from leaves chilledin the light, which were in the uncoupled state, also did notshow a rapid decay, unless an efficient uncoupler such as gramicidinwas added. These results suggest that even a considerable uncouplingof thylakoids, brought about by chilling of leaves in the light,is not sufficient to cause a marked acceleration of the decayof the flash-induced change in absorbance at 518 nm. Therefore,analysis at 518 nm is not always a sensitive method for assessingthe coupling state of thylakoids. (Received July 1, 1991; Accepted October 4, 1991)