The modulation of membrane fluidity by hydrogenation processes. III. The hydrogenation of biomembranes of spinach chloroplasts and a study of the effect of this on photosynthetic electron transport

A method is reported for the in situ modification of the lipids of isolated spinach chloroplast membranes. The technique is based on a direct hydrogenation of the lipid double bonds in the presence of the catalyst, chlorotris(triphenylphosphine)rhodium (I). The pattern of hydrogenation achieved suggests that the catalyst distributes amongst all of the membranes. The polyunsaturated lipids within the membranes are hydrogenated at a faster rate and at an earlier stage than are the monoenoic lipids.Whilst addition of the catalyst to the chloroplast causes an initial 10–20% decrease in Hill activity, saturation of up to 40% of the double bonds present can be accomplished without causing further significant alterations in photosynthetic electron transport processes or marked morphological changes of the chloroplast structure as observed in the electron microscope.     

Modulation of chloroplast membrane lipids by homogeneous catalytic hydrogenation

A method is reported for the modification of lipids in situ in chloroplast membrane by which a homogeneous, water-soluble catalyst Pd(QS)2 (QS, sulphonated alizarine; C14H6O7NaS) is incorporated into the thylakoids of isolated chloroplast. The catalyst itself did not affect the photosynthetic activity but caused an extensive loss of unsaturated fatty acids in the presence of hydrogen gas. The polyunsaturated fatty acids were hydrogenated at a faster rate than the monoenoic acids. During hydrogenation the orientational ordering of membrane lipids, as measured with the C-12 positional isomer of spin-labelled stearic acid, displayed a slight increase in agreement with the alterations in membrane composition. Progressive saturation of double bonds of lipids primarily inhibits electron transport between the photosystems followed by the inhibition of electron flow around photosystem II. Photosystem I electron transport was not inhibited even by 50% fatty acid hydrogenation. We suggest that using Pd(QS)2 catalyst for thylakoid hydrogenation offers an excellent technique to study the role of various unsaturated fatty acids in the regulation of membrane fluidity and photosynthetic processes.     

The modulation of membrane fluidity by hydrogenation processes. II. Homogeneous catalysis and model biomembranes

A homogeneous catalyst, chlorotris (triphenylphosphine) rhodium (I) has been incorporated into model biomembrane structures in the form of lipid bilayer dispersions in water. This enables the hydrogenation of the double bonds of the unsaturated lipids within the bilayers to be accomplished. To decide the optimum conditions for efficient hydrogenation the reaction conditions have been varied. The effect of catalyst concentration, hydrogen gas pressure and lipid composition (with and without cholesterol) have all been studied. The partition of the catalyst into the lipid medium was checked by rhodium analysis. The results show that an increase of catalyst concentration or an increase of hydrogen gas pressure leads to increasing rates of hydrogenation. Successful hydrogenation was accomplished with different types of lipid dispersions (mitochondrial, microsomal and erythrocyte lipids). A selectivity of the homogeneous hydrogenation process is indicated. The polyunsaturated fatty acyl residues are hydrogenated at an earlier stage and at a faster rate than the monoenoic acids. Furthermore, an increase in the proportion of cholesterol to lipid within the bilayer structures causes a progressive decrease in the rate of hydrogenation. The fluidity of the lipid bilayers can be altered to such an extent by the hydrogenation process that new sharp endotherms corresponding to the order-disorder transition of saturated lipids occur at temperatures as high as 319 K. Some potential uses of hydrogenation for the modulation of cell membrane fluidity are discussed as well as the design of new types of catalyst molecules.     

Action of a homogeneous hydrogenation catalyst on living Tetrahymena mimbres cells

Various conditions were tested in an attempt to hydrogenate the unsaturated fatty acids of living Tetrahymena mimbres with the homogeneous catalyst palladium di-(sodium alizarine monosulfonate) without causing serious damage to the cells. Using a low (20 micrograms/ml) catalyst concentration in the external medium, hydrogenation of greater than 20% of surface membrane lipid double bonds were obtained, but hydrogenation of intracellular membranes was minimal. When exposed to H2, cells preincubated with inactive catalyst for several hours and visibly loaded with the catalyst lost viability as soon as hydrogenation exceeded trace levels. Material secreted by Tetrahymena into their medium effectively inhibited hydrogenation of added oleic acid, normally a good substrate. Mucus secreted by the cells, soluble proteins isolated from cell homogenates, bovine serum albumin, and cysteine were also inhibitory, but the inhibition could be overcome by employing higher catalyst concentrations. Although some enzymatic retroconversion of saturated lipids back to unsaturated lipids appeared to take place, the scale of the conversion was small, and further experimentation will be required to understand the mechanism involved. The selective hydrogenation of surface membranes achieved by these methods may be especially useful to those interested in fluidity effects on plasma membrane properties.     

Homogeneous catalytic hydrogenation of the polar lipids of pea chloroplasts in situ and the effects on lipid polymorphism

The pattern of hydrogenation of polar lipids of pea chloroplasts incubated in the presence of the homogeneous catalyst Pd(QS)₂, a sulphonated alizarine complex of Pd(II) has been examined. Analysis of the fatty acyl residues of the major lipid classes from chloroplast suspensions at intervals during incubation under hydrogenating conditions showed that susceptibility to hydrogenation increased in the order monogalactosyldiacylglycerol > digalactosyldiacylglycerol > sulphoquinovosyldiacylglycerol > phosphatidylglycerol. Almost 80% of the total number of double bonds in the polar lipids were removed after 2-h incubation under the conditions employed. The consequence of hydrogenation on the phase behaviour of total polar lipid extracts in aqueous dispersions were examined by freeze-fracture electron microscopy, X-ray diffraction and differential scanning calorimetry. These data indicate that progressive hydrogenation of tne lipids in situ produce a change in the organisation of the lipid when dispersed in water. Single bilayer vesicles are converted to large aggregates of planar bilayer stacks in which the hydrocarbon chains are predominantly in the gel phase configuration. Studies of lipids dispersed in 20 mM MgCl₂ suggest that cohesion between the hydrocarbon chains gradually ameliorates the repulsive effects of the charged lipids, sulphoquinovosyldiacylglycerol and phosphatidylglycerol. This results in the formation of a sheet-like lamellar phase characteristic of dispersions of saturated monogalactosyldiacylglycerols which dominates the total polar lipid extracts of pea chloroplasts.     

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

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.     

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.     

Modulation of membrane fluidity in living protoplasts of Nicotiana plumbaginifolia by catalytic hydrogenation

A homogeneous water-soluble Ru catalyst, has been incorporated into mesophyll protoplasts isolated from Nicotiana plumbaginifolia leaves. In the presence of hydrogen gas this complex causes an extensive loss of unsaturated fatty acid bonds and a concomitant increase in microviscosity of the cellular membranes. Although the gradual reduction of the level of unsaturation, per se, is accompanied by considerable cell damage, there is an optimum reaction time where approximately 50% of the protoplasts are still living and about 20% of the double bounds initially present in fatty acyl residues have undergone hydrogenation. The possible mechanism of the self regulatory process competing with the hydrogenation in the early stages of the reaction is also discussed.     

Effects of the catalytic hydrogenation of microsomal lipids upon four enzyme activities involved in oleic acid desaturation

Catalytical hydrogenation of the unsaturated fatty acyl residues of microsomal lipids was realized for different times. Progress of the reaction was followed by calculating the progressive loss of double-bonds in 100 initial acyl residues (percentage of hydrogenation). The maximum loss observed was 45% after 60 min.The drop in polyunsaturated faty acid content was coupled with an increase in the amount of stearic acid and oleic acid.The order parameter of microsomal lipids, measured by ESR, increased parallely to the reduction of double bonds. Maximum hydrogenation of microsomal lipids strongly (200–250%) stimulated microsomal NADH-ferricyanide reductase activity. NADH-cytochrome c reductase, lysophosphatidylcholine-acyl-transferase and oleoyl-phosphatidylcholine desaturase were inhibited (40%, 100% and 100% respectively). These modifications of enzyme activities are discussed in conjunction with the changes observed in membrane fluidity, following hydrogenation of microsomal lipids     

Increased thermal stability of pigment-protein complexes of pea thylakoids following catalytic hydrogenation of membrane lipids

The membrane lipids of pea thylakoids were hydrogenated in situ using the homogeneous catalyst palladiumdi-(sodium alizazine monosulphonate). Following hydrogenation, particle-free patches corresponding to phase-separated gel-phase lipids were observed in the fracture-faces of thylakoid membranes. Freeze-fracture studies on samples of hydrogenated thylakoids incubated at elevated temperatures indicated that hydrogenation reduces the tendency of the heated membranes to destack and vesiculate at higher temperatures. Measurements of chlorophyll a fluorescence emission and the thermal properties of hydrogenated thylakoids suggest that the hydrogenation process also leads to an increase in the thermal stability of pigment-protein complexes of the Photosystem II light-harvesting apparatus.     

Effect of catalytic hydrogenation of Tetrahymena ciliary phospholipid fatty acids on ciliary phospholipase A activity

Detached Tetrahymena cilia were treated for increasing periods of time with the homogeneous hydrogenation catalyst palladium di(sodium alizarine monosulphonate). This caused a 4-70% reduction in the number of double bonds in phospholipid-bound fatty acids and a concurrent decrease in membrane fluidity as detected by ESR measurements. Ciliary phospholipase A activity was markedly inhibited when as little as 13% of the fatty acid double bonds had been hydrogenated, suggesting that the enzyme activity is very sensitive to changes in membrane fluidity.     

Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C_16:1 and C_18:1 lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.     

THE MOLECULAR ORGANIZATION OF CHLOROPLAST MEMBRANES

The presence of subunits in chloroplast membranes is suggested by polarization, fluorescence, and X-ray studies. Subunits (quantasomes) may be observed in the electron microscope on dried shadowed membranes and in replicas of membranes produced by the freeze-etching technique. Regular subunits are also observed with the electron microscope in thin sections of chloroplast membranes. Chemical considerations suggest that many membranes are composed of lipoprotein subunits. Thin sections reveal two types of chloroplast membranes, the fret membranes composed of one layer of subunits, and the partitions composed of two layers of subunits. Chloroplast membranes consist of about 45% protein and 55% lipid. Some 80% of the lipids are the highly surfactant glycolipids. In this paper the subunits are visualized as assymetric lipoproteins, probably having a protein core surrounded by components determined by the nature and environment of the membrane. Since the stroma, fret channels, and loculi contain aqueous materials, it is further postulated that the membranes bordering these spaces bind the highly surfactant glycolipids. The region between the two rows of subunits in the partition appears to be highly hydrophobic, rich in chlorophyll, and low in glycolipids. Some chlorophyll also may occur within the subunits both in the partitions and in the fret membranes. Since four subunits appear to comprise a quantasome, at least two types of forces, inter- and intra-quantasome forces, bind the subunits together in sheets. Chloroplast membranes thus differ from a “unit membrane” in two important respects: (1) they must be an aggregate of globular subunits, and (2) the lipoprotein subunits consist of a protein matrix which binds the chlorophylls and lipids by hydrophobic association with their hydrocarbon moieties.     

Magnetic resonance studies of dynamic organisation of lipids in chloroplast membranes

Spinach chloroplast membranes and aqueous dispersions of their extracted lipids have been studied by spin label (stearic acid) electron spin resonance and carbon-13 nuclear magnetic resonance techniques. Combined with electron microscope studies, first systematic evidence is found for the existence of a dynamic lipid-bilayer structure in the chloroplast membranes.     

Chloroplast lipid synthesis and lipid trafficking through ER-plastid membrane contact sites

Plant chloroplasts contain an intricate photosynthetic membrane system, the thylakoids, and are surrounded by two envelope membranes at which thylakoid lipids are assembled. The glycoglycerolipids mono- and digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol as well as phosphatidylglycerol, are present in thylakoid membranes, giving them a unique composition. Fatty acids are synthesized in the chloroplast and are either directly assembled into thylakoid lipids at the envelope membranes or exported to the ER (endoplasmic reticulum) for extraplastidic lipid assembly. A fraction of lipid precursors is reimported into the chloroplast for the synthesis of thylakoid lipids. Thus polar lipid assembly in plants requires tight co-ordination between the chloroplast and the ER and necessitates inter-organelle lipid trafficking. In the present paper, we discuss the current knowledge of the export of fatty acids from the chloroplast and the import of chloroplast lipid precursors assembled at the ER. Direct membrane contact sites between the ER and the chloroplast outer envelopes are discussed as possible conduits for lipid transfer.     

The Chloroplast Outer Envelope Membrane： The Edge of Liaht and Excitement

The chloroplast is surrounded by a double-membrane envelope at which proteins, ions, and numerous metabolites including nucleotides, amino acids, fatty acids, and carbohydrates are exchanged between the two aqueous phases, the cytoplasm and the chloroplast stroma. The chloroplast envelope is also the location where the biosynthesis and accumulation of various lipids take place. By contrast to the inner membrane, which contains a number of specific transporters and acts as the permeability barrier, the chloroplast outer membrane has often been considered a passive compartment derived from the phagosomal membrane. However, the presence of galactoglycerolipids and β-barrel membrane proteins support the common origin of the outer membranes of the chloroplast envelope and extant cyanobacteria. Furthermore, recent progress in the field underlines that the chloroplast outer envelope plays important roles not only for translocation of various molecules, but also for regulation of metabolic activities and signaling processes. The chloroplast outer envelope membrane offers various interesting and challenging questions that are relevant to the understanding of organelle biogenesis, plant growth and development, and also membrane biology in general.     

Relationship between Chloroplast Membrane Fatty Acid Composition and Photosynthetic Response to a Chilling Temperature in Four Alfalfa Cultivars

The photosynthetic responses of four alfalfa (Medicago sativa L.) cultivars to 10 and 22 C air temperatures were examined and the relationship between the photosynthetic response at 10 C and the fatty acid composition of the chloroplast membranes was determined. Chilling-resistant cultivars exhibited moderate reductions in photosynthesis at 10 C, compared to 22 C, and contained a significantly greater percentage of polyunsaturated fatty acids in the chloroplast membrane and a greater double bond index than the chilling-sensitive cultivars. The chilling-sensitive cultivars exhibited severe reductions in photosynthesis at 10 C, compared to 22 C. The reduction in photosynthesis at 10 C is shown to be negatively correlated (r = −0.94) with the double bond index of the chloroplast membranes of the cultivars observed.

The results support the hypothesis that reduced photosynthesis due to chilling temperatures is influenced by the unsaturated fatty acid composition of the chloroplast membrane which affect temperature-induced phase changes in chloroplast membrane lipids.

     

Lipid hydrogenation induces elevated 18:1-CoA desaturase activity in Candida lipolytica microsomes.

Microsomal membranes prepared from the mesophilic yeast Candida lipolytica grown at 10 degrees C were hydrogenated by the homogeneous Pd-catalyst, palladium di (sodium alizarine sulfonate) (Pd(QS)2). After hydrogenation to various levels, the microsomes were washed free of the Pd-complex and transferred to a reaction mixture (containing NADH, MgCl2, ATP, CoA and [14C]18:1-CoA) for assay of 18:1-CoA desaturase activity. Microviscosity alterations were also followed by measuring changes in DPH fluorescence polarization. Rapid catalytic hydrogenation of unsaturated fatty acids of the lipids occurred within 20-120 s, resulting in large increases in 16:0, 18:0 and 18:1 acids and decreases in 18:2 acid. In the range 7-20% 18:0 content, a pronounced increase in desaturase activity was observed, with a maximum of greater than 2-fold at a 18:0 content of 12%, followed by a decrease to the initial activity at 33% 18:0 content. These changes were well-correlated with changes in microviscosity, maximal desaturase activity occurring in the DPH fluorescence anisotropy range of 0.23-0.24; above and below this range, desaturase activities were close to the initial control values. It is suggested that the hydrogenation-induced increase in the formation of 18:2 from 18:1-CoA (proceeding partly through direct desaturation of PC) may be due to changes in conformation of the membrane-bound desaturase enzyme complex as a result of controlled rigidification of the surrounding lipids. The operation of such a self-regulating control mechanism would be consistent with a previously proposed model for microsomal desaturase action.     

Heterogeneous asymmetric reactions. Part 24. Heterogeneous catalytic enantioselective hydrogenation of the C==N group over cinchona alkaloid modified palladium catalyst.

The enantioselective hydrogenation of C==N-C group containing compounds over modified metal catalysts is as yet an uninvestigated research area. This work contains results obtained on the hydrogenation of 1-pyrroline-2-carboxylate esters and sodium salt over cinchona alkaloid-modified alumina-supported Pd catalyst. The effect of the reaction parameters and the structure of the alkaloid molecule on hydrogenation rate and enantioselectivity allowed us to assume that on the catalyst surface only a weak interaction exists between the modifier and the substrate, resulting in the low enantiomeric excesses (up to 20%) obtainable in these reactions.     

Free and membrane-bound chloroplast polyribosomes Chlamydomonas reinhardtii.

Over half of the chloroplast ribosomes isolated from growing cultures of Chlamydomonas reinhardtii are bound to chloroplast thylakoid membranes if completion of nascent polypeptide chains is prevented by chloramphenicol. The free chloroplast ribosomes are recovered in homogenate supernatants, and presumably originate from the chloroplast stroma. Only about 10% of these free chloroplast ribosomes are polyribosomes, even under conditions when 70% of free cytoplasm ribosomes are recovered as polyribosomes. The nonionic detergent Nonidet P-40 liberates atypical polyribosomes (Type I), from membranes, which require both ribonuclease and proteases for complete conversion to monomeric ribosomes. Thus Type I particles are held together by mRNA but are also held together by peptide bonds. These Type I polyribosomes probably are not bound to intact membrane, but might be bound to some protein-containing sub-membrane particle. The Type I polyribosomes are dissociated to ribosomal subunits by puromycin and high salt, and contained 0.2 to 1 nascent chain per ribosome. If membranes are treated with Nonidet and proteases at the same time, polyribosomes which are digested to monomeric ribosomes by ribonuclease alone (Type II) are obtained. Type II polyribosomes are smaller than Type I, and probably represent the true size distribution of polyribosomes on the membranes. At least 50% of the membrane-bound ribosomes are polyribosomes, since that much membrane bound chloroplast RNA is recovered as Type I or Type II polyribosomes.