LászlóI. Horváth

Characteristics of Cu deficiency-induced inhibition of photosynthetic electron transport in spinach chloroplasts

Abstract In the present work we studied the effect of Cu deficiency on spinach chloroplasts. We found that in spinach the electron transport was inhibited as reported previously for sugar beet (Droppa, M., Terry, N. and Horvath, G. (1984) Proc. Natl. Acad. Sci. USA (1984) 81, 2369–2373). The breakpoint of the Arrhenius plot of the whole electron-transport activity was shifted from +6°C to +12°C in Cu-deficient chloroplasts. A similar effect could be observed with a spin-labelled probe, when the rotational correlation time was plotted vs. the reciprocal temperatures. This indicates that the membrane fluidity might be changed by Cu deficiency. The lipid/protein ratios were similar in both control and deficient chloroplasts. On the other hand, the saturated/unsaturated ratio of phosphatidylcholine (PC), phosphatidylglycerol (PG) and sulpholipids (SL) was increased but that of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) decreased. We conclude that Cu deficiency does not change the entire membrane fluidity but rather the lipid composition of the microenvironment of some electron-transport components. The inhibition of Photosystem II electron transport in Cu-deficient chloroplasts was characterized by thermoluminescence and 2-dimensional gel electrophoresis. It was found that Cu deficiency shifted the main peak of the glow curve from +18°C to +8°C, similar to that of DCMU-poisoned chloroplasts. Two apoproteins of the 29 kDa polypeptide disappeared in Cu-deficient chloroplasts which indicates that this polypeptide has a regulatory role in ensuring the normal electron flow between Q A and Q B .

Functional significance of the lipid-protein interface in photosynthetic membranes

The functional significance of the lipid-protein interface in photosynthetic membranes, mainly in thylakoids, is reviewed with emphasis on membrane structure and dynamics. The lipid-protein interface is identified primarily by the restricted molecular dynamics of its lipids as compared with the dynamics in the bulk lipid phase of the membrane. In a broad sense, lipid-protein interfaces comprise solvation shell lipids that are weakly associated with the hydrophobic surface of transmembrane proteins but also include lipids that are strongly and specifically bound to membrane proteins or protein assemblies. The relation between protein-associated lipids and the overall fluidity of the thylakoid membrane is discussed. Spin label electron paramagnetic resonance spectroscopy has been identified as the technique of choice to characterize the protein solvation shell in its highly dynamic nature; biochemical and direct structural methods have revealed an increasing number of protein-bound lipids. The structural and functional roles of these protein-bound lipids are mustered, but in most cases they remain to be determined. As suggested by recent data, the interaction of the non-bilayer-forming lipid, monogalactosyldyacilglycerol (MGDG), with the main light-harvesting chlorophyll a/b-binding protein complexes of photosystem-II (LHCII), the most abundant lipid and membrane protein components on earth, play multiple structural and functional roles in developing and mature thylakoid membranes. A brief outlook to future directions concludes this review.

Protoplast plasmalemma fluidity of hardened wheats correlates with frost resistance.

The importance of the physico-chemical state of plant membranes in assuring survival at reduced temperatures has been emphasized [ 11. The thermotropic phase transition temperature of the structural lipids in chilling-sensitive plants is at higher values than in chilling-resistant plants [2,3]. Chillingand frostresistant plants differ in several respects, one of these being their lower temperature limits. While the latter survive, after proper hardening, at rather low temperatures, the former suffer cellular damage at temperatures rather higher than the freezing point of the intracellular water. Extrapolation of the concept of homeoviscous adaptation of membrane fluidity, observed with both prokaryotic [4] and eukaryotic [5] cells, to frost-resistant plants, implies that membrane fluidity is increased in some way during the hardening process. However, senescence induces a decrease in the fluidity of membranes in almost every system studied [6-81. Thus the questions arise: (1) Whether the hardening plants can, in some way, compensate for the effects of senescence; (2) To what extent these opposing trends in membrane fluidity affect the survival of such plants at reduced temperatures. The key membrane involved in developing frost tolerance is the plasmalemma and to gain information about its physico-chemical state protoplasts were isolated from leaves of four cultivars of the wheat, Triticum aestivum L., of different hardiness, in order to follow the changes in microviscosity during hardening. 2. Materials and methods

Complex hydrogenation/oxidation reactions of the water-soluble hydrogenation catalyst palladium Di (sodium alizarinmonosulfonate) and details of homogeneous hydrogenation of lipids in isolated biomembranes and living cells

Palladium di (sodium alizarinmonosulfonate) is a highly efficient catalyst for the hydrogenation of unsaturated fatty acids esterified in lipids of model or biological membranes, enabling the study of the relationship between function and the physical state of membranes. However, the catalyst shows a complex behavior in the action of molecular hydrogen and oxygen, giving rise to the formation of at least four products. Two of these are free radicals. Owing to this complexity, precise control of the reaction requires pretreatment of the catalyst. When partial hydrogenation of the palladium complex is followed by air oxidation, a catalyst solution is produced which is stable on air and maintains catalytic hydrogenation activity for several days. This form of the catalyst induces hydrogenation of unsaturated lipids with no induction period making a strict timing of the procedure possible. Of the several other factors affecting the outcome of membrane hydrogenations, one of the most important is the accessibility to the catalyst of particular membrane regions or lipid pools. Differences in accessibility may arise as a consequence of different local microviscosities or their change during hydrogenation, of the appearance of distinct liquid crystalline phases, and of strong protein-lipid interactions. Obviously, in case of whole-cell hydrogenations, the accessibility is influenced by the spatial separation of the organelles, as well.

Immune-complex-induced transglut aminase activation: Its role in the Fc-receptor-mediated transmembrane effect on peritoneal macrophages

Abstract The binding of soluble immune complexes (IC) or haemolysin-sensitized erythocytes (EA) to the Fc receptor of rat peritoneal macrophages was followed by a rapid increase of macrophage transglutaminase activity measured in cell homogenates and a time-dependent incorporation of 14 C-methylamine into proteins of the intact cells. Methylamine, a competitive substrate inhibitor of transglutaminase could inhibit EA rosette formation as well as the soluble IC- or EA-induced lipid reordering of the plasma membrane of macrophages. Cytochalasin B (CB) which prevents EA rosette formation as well as IC-induced lipid reordering did not affect the stimulation of transglutaminase activity by IC. The possible relation of transglutaminase activation, lipid reordering and the contractile system to each other in the IC (multivalent ligand)-induced Fc receptor redistribution is discussed.

Homogeneous catalytic hydrogenation of lipids in the photosynthetic membrane: Effects on membrane structure and photosynthetic activity

Abstract We have carried out a series of experiments in which the lipid composition of the photosynthetic membrane has been altered by the homogeneous catalytic hydrogenation of the unsaturated fatty acid residues of membrane lipids. The modified membrane was investigated by electron microscopy, electron-spin resonance and fluorescence polarization methods. Alteration in the functional characteristics of the hydrogenated membrane was monitored by the measurement of photophosphorylation and electron-transport activities. The following results were found. (a) Saturation of 10% of the fatty acyl double bonds induced a definite decrease in the dimension of both thylakoids and loculi. Microdensitometry showed that these structural changes arose from a thickening of the single membranes with a simultaneous decrease in the spacing between membranes. These changes might be accounted for by the alignment of the hydrocarbon chains of saturated lipids and the increased hydrophobicity of the membranes. (b) The orientational pattern of chlorophyll- a molecules was not altered by saturating up to 50% of fatty acyl double bonds in membrane lipids, indicating that the energy-transfer processes amongst the chlorophyll molecules remained functional after hydrogenation. (c) Saturation of double bonds of lipids inhibited whole electron transport prior to the inhibition of Photosystem II and Photosystem I activity, which may suggest that the unsaturation level of fatty acids plays a crucial role by ensuring the lateral mobility of plastoquinone between Photosystem II and Photosystem I.

SPIN LABEL EPR STUDY OF LIPID SOLVATION OF SUPRAMOLECULAR PHOTOSYNTHETIC PROTEIN COMPLEXES IN THYLAKOIDS

Lipid-protein association in the chloroplast membrane and its various thylakoid fractions from higher plants, namely pea and maize, rich in Photosystem I (PSI) and Photosystem II (PSII), respectively, were studied using EPR spectroscopy of spin-labelled lipid molecules. All the EPR spectra consisted of two spectral components corresponding to bulk fluid lipids and solvation lipids motionally restricted at the hydrophobic surface of membrane proteins. Spin-labelled stearic acid and phosphatidylglycerol exhibited marked selectivity towards the supramolecular protein complexes of both PSI and PSII although to different extent. In addition, lipid-protein titration experiments are described for partially delipidated PSII-enriched membrane fractions of pea chloroplasts, incorporating unlabelled egg phosphatidylcholine prior to or after the incorporation of spin-labelled lipids. Two sets of solvation sites were resolved by timed labelling experiments and a significant result of these studies was that a well-defined population of solvation sites (approx. 100 mol lipids/820 kDa protein) was rapidly exchanged by laterally diffusing membrane lipids, while other solvation sites (approx. 50 mol lipids/820 kDa protein) were exchanged much slower or not exchanged at all.

Effect of choline chloride on fatty acid chain ordering in membranes of wheat (Triticum aestvium L. cv. Miranovskaja 808)

A winter variety (Miranovskaja 808) of wheat (Triticum aestivum L.) was grown in water cultures containing 0, 5, 15, 30, and 60 mM choline chloride. There was an increase in the phosphatidylcholine level at the expense of phosphatidic acid, parallel with an increasing choline concentration in the cultivating medium. While the ratio of free sterols to phospholipids remained essentially constant in the seedlings, there was an increase in the ratio of saturated-to-unsaturated fatty acids of the phospholipid fraction. Probing the protoplasts obtained from the leaves of the seedlings, with spin labeled 5-doxyl- and 16-doxyl stearic acids, indicated a progressive rigidifying of the hydrophobic core of the plasmalemma. It is suggested that this is a manifestation of compensatory mechanisms by which plants attempt to maintain unchanged average membrane fluidity across their membranes in response to the fluidizing effect of choline head groups.

Relation of Raman order parameters to spin labeling parameters

For the quantitation of Raman and spin labeling data order parameters are commonly used. The spin label order parameter measured at any depth in the layer is a weighed sum of the segmental order since, due to fast conformational interconversions, each CH2 segment is partly in trans and partly in non-trans, e.g. gauche, kink, jog, etc. conformation during the measurement. The weighing factor, the trans finding probability, varies along the chain (cf. flexibility profile) but its mean value should be equal to the Raman trans order parameter. This correlation is illustrated with the experimental data obtained for dipalmitoyl phosphatidylcholine and n-alcohol mixtures. The rate of rotational diffusion, a dynamical parameter from spin labeling studies, is correlated with the lateral packing density as measured by the Raman lateral order parameter. For the obtained linear correlation a qualitative explanation is given. The effect of a series of long chain alcohols on the phase transition characteristics of dipalmitoyl phosphatidylcholine was investigated. The possible role of hydrogen bonding in the interfacial region is emphasized.

Phospholipase C digestion of rat liver plasma membrane stimulates adenylate cyclase.

Abstract Brief treatment of rat liver plasma membranes with phospholipase C of Clostridium welchii increased both the ratio of saturated to unsaturated fatty acids and the ratio of cholesterol to phospholipids. Using 5-doxylstearic acid spin probes two breaks at 29 and 19.6 °C could be observed in the order parameter, S A , vs temperature curve for untreated membranes. Upon phospholipase C digestion the lower phase transition temperature was shifted to 23 °C, while the higher phase transition temperature could not be detected up to 40 °C. The order parameter, S A , was consistently higher at all temperatures in the phospholipase C-treated membranes. As phospholipase C is known to attack the outer lamella, these results can be interpreted as indicating an increase in ordering (i.e., decrease in fluidity) of the outer membrane lamella. On the other hand, an increase in basal activity of adenylate cyclase of the treated membranes was observed with an apparent reduction of the activation energies both below and above the break (at 20 °C) in the Arrhenius plot of enzyme activity. Phospholipase C treatment did not affect the temperature of the break in Arrhenius kinetics of the enzyme. The results are discussed in terms of the role of the ordering state of membrane lipids in adenylate cyclase activity.