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Monogalactosyldiacylglycerol: The most abundant polar lipid in nature
378
TIBS - October 1983
Monogalactosyldiacylglycerol: the most abundant polar lipid in Nature Kleoniki Gounaris and James Barber Lipids which do not form bilayers exist in most biological membranes but not as profusely as in the chloroplast thylakoids of oxygen-evolving photosynthetic organisms. Almost half the polar lipid content o f this membrane is monogalactosyldiacylglycerol which does not form bilayers. The function of this unusual lipid with its lack o f electrical charge and high degree of unsaturation is to provide a fluid environment in which the diffusional processes of photosynthetic electron transfer can occur and, maybe, to facilitate the optimal packing of large intrinsic proteins within the naturally occurring bilayer structure. The most extensive membrane found in the biosphere is the thylakoid lamellae system of oxygen evolving photosynthetic organisms. Approximately 40% of the dry weight of this membrane consists of polar lipids, of which about half is restricted to the one lipid class, monogalactosyldiacylglycerol (MGDG) (see Table I). It follows, therefore, that this neutral galactolipid must be the most abundant polar lipid in Nature. Presumably it has been selected for chemical and physical properties which facilitate the functional and structural requirements of the photosynthetic membrane. However, when isolated and purified it has unusual properties which, at first sight, are difficult to reconcile with the lipoprotein bilayer structure from which it originates. Kleoniki Gounaris and James Barber are members of the ARC Photosynthesis Research Group in the Department of Pure and Applied Biology, Imperial College, London SW7 2BB, UK. cm$'rR~¢1",o*/
Occurrence and distribution MGDG was first isolated from wheat flour and characterized as a galactose containing lipid by Carter I in 1956. Subsequently it was established that the glycerol residue in the glycolipid had the D configuration '2 and the chemical structure was defined as 1,2-diglyceride with the galactose moiety attached in the C-3 position 3. Isolation of the pure monogalactolipid by Sastry and Kates ~ from runner bean leaves established that it was identical to that occurring in wheat flour. Its chemical structure is described as 1,2 diacyl-3-O(/3-Dgalactopyranosyl)-sn-glycerol and is shown diagrammatically in Fig. 1. Since its discovery it has been identified in all plant tissue and algae investigated. However, although this lipid predominates in the thylakoid membrane it only comprises a minor component of other membrane systems. Moreover, even then it is restricted to membranes which are closely
related to the thylakoid, namely the chloroplast envelope and plastid membranes of non-photosynthetic tissue. In the case of the chloroplast envelope membranes, unlike the thylakoid, the amount of digalactosyldiacylglycerol (DGDG) exceeds that of the monogalactosyl lipid5. The significance of the difference in the proportions of monoand digalactolipid, or indeed of other polar lipids, occurring in the two types of membranes of mature chloroplasts is unclear, but presumably it reflects their quite different functional properties. The polar lipids of the thylakoids are characterized by a strikingly high number of double bonds in their acyl chains, MGDG being the major contributor to this unsaturation. Indeed, studies of the molecular nature of MGDG isolated from the thylakoids of a wide range of plants have indicated that the 1-1inolenoyl/2-1inolenoyl (di-Cl8:3) species predominates and in some cases accounts for as much as 90% of the total content of this lipid class". More saturated fatty acid residues of MGDG do occur in some photosynthetic organisms, TABLE 1. A typical lipid class composition of spinach thylakoid membranes Lipid class
% mol
Monogalactosyldiacylglycerol (MGDG) Digalactosyldiacylglycerol
48
Phosphatidylglycerol Sulphoquinovosyldiacylglycerol (SQDG) Phosphatidyl choline
8 2
(PC) Other phospholipids
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notably the blue-green algae (cyanobacteria) but this is an exception to the general rule. Such a high degree of unsaturation in a membrane, the functional activities of which require exposure to light and involve the production of molecular oxygen, is surprising since such conditions would favour oxidation of unsaturated fatty acids.
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Properties and structure in aqueous systems A number of physical techniques have been used to examine the properties of isolated MGDG dispersed in an aqueous environment. In 1973 Shipley et al. ~ reported the results of an X-ray diffraction study of the phase behaviour of MGDG over the concentration range C = 1.0-0.5 (where C = g iipid/g lipid + water) and the temperature range 80 to - 15°C. This work established that under these conditions the lipid forms a hexagonal phase, with water being incorporated only up to the concentration where C = 0.78. Above this concentration the lipid organization was insensitive to addition of more water, with water and the hydrated MGDG co-existing as separate phases. With increasing hydration the dimension of the phase increased, reaching a limiting diameter of 6.25 nm at 0°C. A diffuse X-ray diffraction line in the wide-angle region at 0.46 nm, at all concentrations, confirmed that the phase was liquid crystalline. From calculations of the lattice parameter it was concluded that of the two possible arrangements for the hexagonal structures, type I and II phases, MGDG forms the latter. Adopting the nomenclature of Luzzati et al.8, such an arrangement consists of water cylinders in a lipid matrix with the hydrocarbon chains radially orientated outwards from the central axis of the cylinders, as depicted in Fig. 2. Low angle and wide-angle X-ray diffraction studies performed on the 1,2dilinolenoyl and 1,2-distearoyl derivatives of MGDG indicate that although the unsaturated species forms the hexagonal type II phase, the fully saturated derivative forms a lamellar structureL The sensitivity of the hexagonal phase on the degree of unsaturation of the fatty acyl residues of MGDG has been demonstrated by subjecting the lipid to different extents of catalytic hydrogenation 1°. These studies indicated that a reduction of an average of 0.5 double
" - "
Fig. 2. Schematic representation of the hexagonal type-ll phase adopted by isolated monogalactosyldiacylglycerol on hydration.
bonds per lipid molecule is sufficient to induce the formation of a lamellar structure, As Fig. 3 shows, freeze-fracture electron microscopy can distinguish clearly between the two phases and it is worth noting that the bilayer induced by hydrogenation is not of the conventional liposomal type but appears to be open flat sheets of lamellae, In its fully unsaturated form, MGDG does not undergo a transition to the gel phase until the temperature falls to - 30°C. It has been found, however, that only a slight reduction in the number of double
bonds per lipid molecule causes transitions to the gel phase to occur above room ternperature TM. The head group of MGDG is small in comparison with the volume of its highly unsaturated fatty acyl chains. This uncharged galactose head group is difficult to hydrate but nevertheless, the lipid will spread from a solvent on an air-water interface to form stable monolayers. The surface-active properties of MGDG, isolated from different photosynthetic tissue and varying in degree of unsaturation, have
Fig. 3. Electronmicrographs of freeze--fracture replicas obtained from monogalactosyldiacylglycerol in (a) its native unsaturated.form and (b) hydrogenated in the presence of Adams" catalyst, to an average number of double bonds per lipid molecule of 2.80.
380 been examinedal. It appears that a strong intermolecular attraction exists between the MGDG head groups since this lipid forms more condensed films than phospholipids with corresponding degrees of unsaturation. Role in the membrane in vivo Organization, phase changes a n d specific interactions. The ability of the
naturally occurring MGDG to form a hexagonal phase not only depends on temperature and degree of fatty acid unsaturation, but also on the presence of other lipids and the proportion in which it is present in the mixtures r2. Besides the pure hexagonal phase, a number of other non-bilayer structures have been observed in the total polar lipid extracts of higher plant thylakoids in the presence of cations TM. However, nonbilayer structures of the type adopted by MGDG or indeed of the type observed in lipid mixtures are not normally observed in the thylakoid membrane, although they can be induced under certain conditions~4. The formation of a bilayer lipid matrixin vivo is thus difficult to reconcile with the tendency of MGDG to adopt non-bilayer configurations and it raises questions concerning the possible role of this unusual lipid in the organization and functional properties of the chloroplast membrane. Based on theoretical considerations Israelachvili et al. 15 have suggested that lipid molecules like MGDG, which have a molecular shape like a cone due to their small head group, may act to pack large protein complexes into biological membranes. Such a function would probably prevent MGDG from adopting non-bilayer structures through lipid-protein interactions. The above authors have suggested further that lipids of such a molecular shape could also stabilize regions of high membrane curvature, an argument specifically applied to the thylakoid membrane by Murphy ~. Because of the sensitivity of the structural properties of MGDG to the number of double bonds in its fatty acid chains, a full description of the role of this lipid in chloroplast thylakoid organization must take account of its high level of unsaturation. Moreover, this highly unsaturated character favours the maintenance of a liquid crystalline lipid matrix even down to -
-
2 0 ° C 17.
Recent studies of fractionated thylakoids indicate that MGDG is more prominent in the appressed granal membranes than in stromal membranesTM, although an absolute lateral asymmetry, as for protein distribution, was not observed TM. A general role for MGDG in packaging large protein complexes in the membrane is supported by this observation since the protein to lipid ratio is higher in appressed than stromal mem-
T I B S - October 1983
branes. Studies on the transverse distribution of galactolipids in thylakoidsz° have indicated that approximately 80% of the MGDG is located in the intra-thylakoid layer of the membrane. Recent reports, however, suggest that both the mono- and digalactosyl lipids are asymmetrically distributed across the thylakoid membrane, the asymmetry being less prominent for MGDG2L A specific function for unsaturated MGDG, which probably involves optimal protein packaging, has been reported by Siefermann-Harmset al. ,~2They have given evidence that it is required for facilitating the close association of chlorophyll a/b light-harvesting protein with the photosystem 2 reaction centre complex so that efficient energy transfer can occur. Also, evidence is emerging that unsaturated MGDG, but not saturated MGDG, is required for the full activity of the CFo--CF~-ATP synthetase system of the thylakoids (Pick, U., Gounaris, K., Barber, J. and Admon, A., unpublished observations). It is interesting to note that the inner mitochondrial membrane, in which a related ATP synthetase system is found, also contains non-bilayerlipids such as cardiolipin which could possibly be required for the organization and activity of this membrane system. Fluidity. The high degree of unsaturation of the acyl chains of MGDG and of the other thylakoid lipids indicates that the membrane is likely to be highly fluid in vivo. Studies with total lipid extracts using the time-dependent fluorescence anisotropy characteristics of the hydrophobic probe, diphenyl hexatriene (DPH), support this contention and give microviscosities of 0.21 poise and a half-angle for the probe rotation of 79° at 25°C (Ref. 23). However, the membrane is a lipoprotein structure in vivo and the presence of protein will not only tend to stabilize the bilayer but will have the effect of ordering the acyl chains of the thylakoid lipids thus reducing the overall fluidity of the bilayer. DPH measurements indicate that in isolated thylakoid membranes at 25°C, the viscosity of the lipid matrix is 0.34 poise while the half angle for the probe's rotation decreases to 48°C (Millner, P. A., Mitchell, R. A. C., Chapman, D. J. and Barber, J., unpublished observations). Even though the presence of proteins brings about an ordering effect, the thylakoid membrane is a relatively fluid system when compared with many other biological membranes. This characteristic fluidity seems to be essential for photosynthetic processes involving lateral, rotational and transmembrane diffusionlT.~L For example, it has recently become clear that photosys-
tems one (PSI) and two (PS2) are laterally separated in the plane of the membrane with PSI located in non-appressed and PS2 in appressed regions17.1L Since these two photosystems have to interact in order to transfer electrons from water to NADP, long range electron transfer must occur. The most likely candidate for this function is the hydrophobic redox carrier, plastoquinone, whose lateral diffusion and ability to transfer electrons to PSI via the cytochrome b6---fcomplex 17.19, is likely to be dependent on the fluidity of the lipid matrix (Olsen, L. F. and Haehnel, W., unpublished observations). Interestingly, the mechanism of regulating optimal fluidity in higher plant thylakoids does not seem to involve significant changes in levels of unsaturation of lipids but rather, the adaptation proceeds by varying the protein to lipid ratio24. Such a finding is not surprising when one considers how sensitive the properties of MGDG are to its degree of unsaturation. If changes in the unsaturation level were to occur, however slight, then it is probable that significant alterations in the structural organization and phase properties of the membrane would follow, which would be detrimental to its functions. Clearly MGDG is an intriguing component of the thylakoids and further studies to establish its interactions with other lipids and membrane proteins will be of considerable importance for elucidating the molecular processes of photosynthesis.
References 1 Carter, H. E., McCluer, R. H. and Slifer, E. D. (1956)J. Am. Chem. Soc. 78, 3735-3738 2 Wickberg, B. (1958) Acta Chem. Scand. 12, 1187-1190 3 Carter, H. E., Hen&y, R. A. and Stanacev, N. Z. (1961)J. Lipid Res. 2,223-227 4 Sastry, P. S. and Kates, M. (1964) Biochemistry 3, 1271-1280 5 Douce, R. and Joyard, J. (1980) in The Biochemistry of Plants, Vol. 4 (Stumpf, P. K., ed.), pp. 321-362, Academic Press 6 Mackender, R. O. and Leech, R. M. (1974) Pl. Physiol. 53,496--502 7 Shipley, G. G., Green, J. P. and Nichols, B. W. (1973 ) Biochim. Biophys. Acta 311,531-544 8 Luzzati, V., Gulik-Krzywicki, T., Rivas, E., Reiss-Husson, F. and Rand, R. P. (1968)J. Gen. Physiol. 5 l, 37-43 9 Sen, A., Williams, W P. and Quinn, P. J. (1981) Biochim. Biophys. Acta 663,380-389 10 Gounaris, K., Mannock, D. A., Sen, A., Brain, A. P. R., Williams, W. P. and Quinn, P. J. (1983) Biochim. Biophys. Acta 732, 229-242 11 Bishop, D. G., Kenrick, J. R., Bayston, J. H., Macpherson, A. S. and Johns, S. R. (1980) Biochim. Biophys. Acta 602, 248-259 12 Quinn, P. J. and Williams, W. P. (1983) Biochim. Biophys. Acta 737, 223-266 13 Gounaris, K., Sen, A., Bmm, A. P. R., Quinn, P. J. and Williams, W. P. (1983) Biochim. Biophys• Acta 728, 129-139
381
T1BS - October 1983 14 Gounaris, K., Brain, A. P. R., Quinn, P. J. and Williams, W. P. (1983)FEBS Lett. 153, 47-51 15 lsraelachvili, J. N., Marcelja, S. and Horn, R. G. (1980)Q. Rev. Biophys. 13, 121-200 16 Murphy, D. J. (1982)FEBS Lett. 150, 19-26 17 Barber, J. (1983) Plant, Cell and Environ. 6, 311-322 18 Gounaris, K., Sundby, C., Andersson, B. and
Barber, J. (1983)FEBSLett. 156, 171)-174 19 Anderson, J. M. and Andersson, B. (1982) Trends Biochem. Sci. 7,288-292 20 Uniu, M. D. and Hat'wood, J. L. (1982) in Developments in Plant Biology, Vol. 8 (Wintermans, J. F. G. M. and Kiiiper, P. J. C., eds.), pp. 359-362, Elsevier Biomedical Press 21 Sundby, C. and Larsson, C. 6th International
Congress in Photosynthesis (in press) 22 Siefemmnn-Harms, D., Ross, J. W., Kaneshiro, K. H. and Yamamoto, H. Y. (1982) FEBS Lett. 149, 191-196 23 Ford, R. C. and Barber, J. (1983) Biochim. BIOphys. Acta 722, 341-348 24 Chapman, D. J., DeFelice, J. G. and Barber, J. (1983) Plant Physiol. 72, 225-228
50 Years Ago then advances in this field have been enormous, mainly because of improved techniques and equipment. The work of Hans Fischer and his colleagues (and their Charles H. Gray contemporaries) was of the highest importFifty years ago, knowledge of the bile pig- jaundice, ethanol had to be added for the ance. Their techniques were those of clasments was limited to a few compounds coiour reaction to take place (the so-called sical organic chemistry - isolation, purifiindirect reaction); in obstructive jaundice cation, elemental analysis to provide an which had been isolated from natural sources; their chemical nature was eluci- the red colour developed without the addi- empirical formula, determination of dated by the methods of classical chemis- tion of ethanol (the direct reaction). Many molecular weight, derivation and degradablood specimens gave an intermediate reac- tion were all necessary preliminaries to try. Early in the nineteenth century, Tiedemann and Gmelin had shown that the tion and were presumed to contain both indicate the structure to be confh-med by synthesis. The system of organic microdirect and indirect bilirubin. yellow pigment in bile and serum and urine By the 1920s Kiister had recognized that analysis developed by Pregl was important of jaundiced subjects was dehydrogenated to green, blue, violet, red, orange and yel- the structure of bilirubin is closely related in this field. The monograph by Fischer and low pigments by nitric acid containing a to that of haem, the prosthetic group of Orth, published in 1937, 3 is a monument to trace of nitrous acid. These changes were haemoglobin; however, the structure of their contribution to the classical work in later found to be due to the formation from bilirubin was not finally elucidated until the bile pigment field. The bilene urobilin 1933, shortly after the structure of haem which has two ethyl groups instead of vinyl bilirubin of green biliverdins, blue and violet biliviolins, red bilirhodins and other itself had been established. Bilirubin was groups was synthesized in 1936 by Siedel coloured oxidation products. Before the shown to be a linear tetrapyrrole and haem a and MeieP, who were both pupils of Hans end of the nineteenth century, bilirubin had cyclic tetrapyrrole; the groups in the side Fischer. The synthesis of bilirubin (a bilidiene) was not accomplished until 1942 been isolated from gallstones and the chains were identical in nature and order2. The above is a brief account of what was by Fischer and PlieningeP. This was a fororange-red pigments urobilin and stercobilin had been found in pathological urine and known of the bile pigments in 1933. Since midable task because of the vinyl groups in two of the r-positions of the four pyrrole in faeces, respectively. Urobilinogen, a rings, and the asymmetry of its linear struccolourless precursor of urobilin in pathologture. ical urines, was shown in 1911 to be identiClassical separation techniques have cal to mesobilirubinogen formed by amalbeen replaced by column, paper, thin layer gam reduction of bilirubin. Biliverdin was and especially high performance high isolated from dog placentae by Lemberg pressure) liquid chromatography. The and Barcroft in 1932. crude polarimeters, colorimeters and specThe early view that bile pigment was troscopes have been superseded by formed in the liver was shown conclusively automated equipment which can record in a to be wrong1; bile pigments are formed in few minutes measurements which in 1933 the cells of the reticuloendothelial system would have taken a day or more to comand carried in the blood to the liver for plete, if they could have been carried out at excretion in the bile. In 1933, jaundice had all. Spectrophotometers for the ultraviolet long been recognized to be due to an and infrared wavelengths of radiation increased concentration of bilirubin in the became available after 1933 and such blood, either as a result of excessive equipment now can record the absorption haemolysis of red blood cells in the curve as a printout or even plot a series of reticuloendothelial system (retention jauncurves o n l i n e for the effluents obtained by dice) or obstruction in the biliary passages HPLC. Improved polarimetry, and mass by stone or tumour (obstructive jaundice). spectrometry, measurement of circular So-called catarrhal jaundice was believed to dichroism, X-ray diffraction and nuclear be caused by obstruction of the bile ducts by ~: magnetic resonance investigations have catarrh. Colorimetric tests for bile pigments revolutionized our knowledge of the in body fluids had been developed and the detailed structure of the bile pigments. diazo reaction was applied by van den Of major importance was the discovery Bergh to provide a quantitative estimation Reproduced by permission of P. A. Norstedt pubin 1943 by Dible, McMichael and Sherof bilirubin in plasma or serum. In retention lishers, Stockholm.
The bile pigments 1933-1983
© 1983.ElsevierSciencePublishersB.V, Amsterdam 0376 5067/83/$O1.00
TIBS - October 1983
Monogalactosyldiacylglycerol: the most abundant polar lipid in Nature Kleoniki Gounaris and James Barber Lipids which do not form bilayers exist in most biological membranes but not as profusely as in the chloroplast thylakoids of oxygen-evolving photosynthetic organisms. Almost half the polar lipid content o f this membrane is monogalactosyldiacylglycerol which does not form bilayers. The function of this unusual lipid with its lack o f electrical charge and high degree of unsaturation is to provide a fluid environment in which the diffusional processes of photosynthetic electron transfer can occur and, maybe, to facilitate the optimal packing of large intrinsic proteins within the naturally occurring bilayer structure. The most extensive membrane found in the biosphere is the thylakoid lamellae system of oxygen evolving photosynthetic organisms. Approximately 40% of the dry weight of this membrane consists of polar lipids, of which about half is restricted to the one lipid class, monogalactosyldiacylglycerol (MGDG) (see Table I). It follows, therefore, that this neutral galactolipid must be the most abundant polar lipid in Nature. Presumably it has been selected for chemical and physical properties which facilitate the functional and structural requirements of the photosynthetic membrane. However, when isolated and purified it has unusual properties which, at first sight, are difficult to reconcile with the lipoprotein bilayer structure from which it originates. Kleoniki Gounaris and James Barber are members of the ARC Photosynthesis Research Group in the Department of Pure and Applied Biology, Imperial College, London SW7 2BB, UK. cm$'rR~¢1",o*/
Occurrence and distribution MGDG was first isolated from wheat flour and characterized as a galactose containing lipid by Carter I in 1956. Subsequently it was established that the glycerol residue in the glycolipid had the D configuration '2 and the chemical structure was defined as 1,2-diglyceride with the galactose moiety attached in the C-3 position 3. Isolation of the pure monogalactolipid by Sastry and Kates ~ from runner bean leaves established that it was identical to that occurring in wheat flour. Its chemical structure is described as 1,2 diacyl-3-O(/3-Dgalactopyranosyl)-sn-glycerol and is shown diagrammatically in Fig. 1. Since its discovery it has been identified in all plant tissue and algae investigated. However, although this lipid predominates in the thylakoid membrane it only comprises a minor component of other membrane systems. Moreover, even then it is restricted to membranes which are closely
related to the thylakoid, namely the chloroplast envelope and plastid membranes of non-photosynthetic tissue. In the case of the chloroplast envelope membranes, unlike the thylakoid, the amount of digalactosyldiacylglycerol (DGDG) exceeds that of the monogalactosyl lipid5. The significance of the difference in the proportions of monoand digalactolipid, or indeed of other polar lipids, occurring in the two types of membranes of mature chloroplasts is unclear, but presumably it reflects their quite different functional properties. The polar lipids of the thylakoids are characterized by a strikingly high number of double bonds in their acyl chains, MGDG being the major contributor to this unsaturation. Indeed, studies of the molecular nature of MGDG isolated from the thylakoids of a wide range of plants have indicated that the 1-1inolenoyl/2-1inolenoyl (di-Cl8:3) species predominates and in some cases accounts for as much as 90% of the total content of this lipid class". More saturated fatty acid residues of MGDG do occur in some photosynthetic organisms, TABLE 1. A typical lipid class composition of spinach thylakoid membranes Lipid class
% mol
Monogalactosyldiacylglycerol (MGDG) Digalactosyldiacylglycerol
48
Phosphatidylglycerol Sulphoquinovosyldiacylglycerol (SQDG) Phosphatidyl choline
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Properties and structure in aqueous systems A number of physical techniques have been used to examine the properties of isolated MGDG dispersed in an aqueous environment. In 1973 Shipley et al. ~ reported the results of an X-ray diffraction study of the phase behaviour of MGDG over the concentration range C = 1.0-0.5 (where C = g iipid/g lipid + water) and the temperature range 80 to - 15°C. This work established that under these conditions the lipid forms a hexagonal phase, with water being incorporated only up to the concentration where C = 0.78. Above this concentration the lipid organization was insensitive to addition of more water, with water and the hydrated MGDG co-existing as separate phases. With increasing hydration the dimension of the phase increased, reaching a limiting diameter of 6.25 nm at 0°C. A diffuse X-ray diffraction line in the wide-angle region at 0.46 nm, at all concentrations, confirmed that the phase was liquid crystalline. From calculations of the lattice parameter it was concluded that of the two possible arrangements for the hexagonal structures, type I and II phases, MGDG forms the latter. Adopting the nomenclature of Luzzati et al.8, such an arrangement consists of water cylinders in a lipid matrix with the hydrocarbon chains radially orientated outwards from the central axis of the cylinders, as depicted in Fig. 2. Low angle and wide-angle X-ray diffraction studies performed on the 1,2dilinolenoyl and 1,2-distearoyl derivatives of MGDG indicate that although the unsaturated species forms the hexagonal type II phase, the fully saturated derivative forms a lamellar structureL The sensitivity of the hexagonal phase on the degree of unsaturation of the fatty acyl residues of MGDG has been demonstrated by subjecting the lipid to different extents of catalytic hydrogenation 1°. These studies indicated that a reduction of an average of 0.5 double
" - "
Fig. 2. Schematic representation of the hexagonal type-ll phase adopted by isolated monogalactosyldiacylglycerol on hydration.
bonds per lipid molecule is sufficient to induce the formation of a lamellar structure, As Fig. 3 shows, freeze-fracture electron microscopy can distinguish clearly between the two phases and it is worth noting that the bilayer induced by hydrogenation is not of the conventional liposomal type but appears to be open flat sheets of lamellae, In its fully unsaturated form, MGDG does not undergo a transition to the gel phase until the temperature falls to - 30°C. It has been found, however, that only a slight reduction in the number of double
bonds per lipid molecule causes transitions to the gel phase to occur above room ternperature TM. The head group of MGDG is small in comparison with the volume of its highly unsaturated fatty acyl chains. This uncharged galactose head group is difficult to hydrate but nevertheless, the lipid will spread from a solvent on an air-water interface to form stable monolayers. The surface-active properties of MGDG, isolated from different photosynthetic tissue and varying in degree of unsaturation, have
Fig. 3. Electronmicrographs of freeze--fracture replicas obtained from monogalactosyldiacylglycerol in (a) its native unsaturated.form and (b) hydrogenated in the presence of Adams" catalyst, to an average number of double bonds per lipid molecule of 2.80.
380 been examinedal. It appears that a strong intermolecular attraction exists between the MGDG head groups since this lipid forms more condensed films than phospholipids with corresponding degrees of unsaturation. Role in the membrane in vivo Organization, phase changes a n d specific interactions. The ability of the
naturally occurring MGDG to form a hexagonal phase not only depends on temperature and degree of fatty acid unsaturation, but also on the presence of other lipids and the proportion in which it is present in the mixtures r2. Besides the pure hexagonal phase, a number of other non-bilayer structures have been observed in the total polar lipid extracts of higher plant thylakoids in the presence of cations TM. However, nonbilayer structures of the type adopted by MGDG or indeed of the type observed in lipid mixtures are not normally observed in the thylakoid membrane, although they can be induced under certain conditions~4. The formation of a bilayer lipid matrixin vivo is thus difficult to reconcile with the tendency of MGDG to adopt non-bilayer configurations and it raises questions concerning the possible role of this unusual lipid in the organization and functional properties of the chloroplast membrane. Based on theoretical considerations Israelachvili et al. 15 have suggested that lipid molecules like MGDG, which have a molecular shape like a cone due to their small head group, may act to pack large protein complexes into biological membranes. Such a function would probably prevent MGDG from adopting non-bilayer structures through lipid-protein interactions. The above authors have suggested further that lipids of such a molecular shape could also stabilize regions of high membrane curvature, an argument specifically applied to the thylakoid membrane by Murphy ~. Because of the sensitivity of the structural properties of MGDG to the number of double bonds in its fatty acid chains, a full description of the role of this lipid in chloroplast thylakoid organization must take account of its high level of unsaturation. Moreover, this highly unsaturated character favours the maintenance of a liquid crystalline lipid matrix even down to -
-
2 0 ° C 17.
Recent studies of fractionated thylakoids indicate that MGDG is more prominent in the appressed granal membranes than in stromal membranesTM, although an absolute lateral asymmetry, as for protein distribution, was not observed TM. A general role for MGDG in packaging large protein complexes in the membrane is supported by this observation since the protein to lipid ratio is higher in appressed than stromal mem-
T I B S - October 1983
branes. Studies on the transverse distribution of galactolipids in thylakoidsz° have indicated that approximately 80% of the MGDG is located in the intra-thylakoid layer of the membrane. Recent reports, however, suggest that both the mono- and digalactosyl lipids are asymmetrically distributed across the thylakoid membrane, the asymmetry being less prominent for MGDG2L A specific function for unsaturated MGDG, which probably involves optimal protein packaging, has been reported by Siefermann-Harmset al. ,~2They have given evidence that it is required for facilitating the close association of chlorophyll a/b light-harvesting protein with the photosystem 2 reaction centre complex so that efficient energy transfer can occur. Also, evidence is emerging that unsaturated MGDG, but not saturated MGDG, is required for the full activity of the CFo--CF~-ATP synthetase system of the thylakoids (Pick, U., Gounaris, K., Barber, J. and Admon, A., unpublished observations). It is interesting to note that the inner mitochondrial membrane, in which a related ATP synthetase system is found, also contains non-bilayerlipids such as cardiolipin which could possibly be required for the organization and activity of this membrane system. Fluidity. The high degree of unsaturation of the acyl chains of MGDG and of the other thylakoid lipids indicates that the membrane is likely to be highly fluid in vivo. Studies with total lipid extracts using the time-dependent fluorescence anisotropy characteristics of the hydrophobic probe, diphenyl hexatriene (DPH), support this contention and give microviscosities of 0.21 poise and a half-angle for the probe rotation of 79° at 25°C (Ref. 23). However, the membrane is a lipoprotein structure in vivo and the presence of protein will not only tend to stabilize the bilayer but will have the effect of ordering the acyl chains of the thylakoid lipids thus reducing the overall fluidity of the bilayer. DPH measurements indicate that in isolated thylakoid membranes at 25°C, the viscosity of the lipid matrix is 0.34 poise while the half angle for the probe's rotation decreases to 48°C (Millner, P. A., Mitchell, R. A. C., Chapman, D. J. and Barber, J., unpublished observations). Even though the presence of proteins brings about an ordering effect, the thylakoid membrane is a relatively fluid system when compared with many other biological membranes. This characteristic fluidity seems to be essential for photosynthetic processes involving lateral, rotational and transmembrane diffusionlT.~L For example, it has recently become clear that photosys-
tems one (PSI) and two (PS2) are laterally separated in the plane of the membrane with PSI located in non-appressed and PS2 in appressed regions17.1L Since these two photosystems have to interact in order to transfer electrons from water to NADP, long range electron transfer must occur. The most likely candidate for this function is the hydrophobic redox carrier, plastoquinone, whose lateral diffusion and ability to transfer electrons to PSI via the cytochrome b6---fcomplex 17.19, is likely to be dependent on the fluidity of the lipid matrix (Olsen, L. F. and Haehnel, W., unpublished observations). Interestingly, the mechanism of regulating optimal fluidity in higher plant thylakoids does not seem to involve significant changes in levels of unsaturation of lipids but rather, the adaptation proceeds by varying the protein to lipid ratio24. Such a finding is not surprising when one considers how sensitive the properties of MGDG are to its degree of unsaturation. If changes in the unsaturation level were to occur, however slight, then it is probable that significant alterations in the structural organization and phase properties of the membrane would follow, which would be detrimental to its functions. Clearly MGDG is an intriguing component of the thylakoids and further studies to establish its interactions with other lipids and membrane proteins will be of considerable importance for elucidating the molecular processes of photosynthesis.
References 1 Carter, H. E., McCluer, R. H. and Slifer, E. D. (1956)J. Am. Chem. Soc. 78, 3735-3738 2 Wickberg, B. (1958) Acta Chem. Scand. 12, 1187-1190 3 Carter, H. E., Hen&y, R. A. and Stanacev, N. Z. (1961)J. Lipid Res. 2,223-227 4 Sastry, P. S. and Kates, M. (1964) Biochemistry 3, 1271-1280 5 Douce, R. and Joyard, J. (1980) in The Biochemistry of Plants, Vol. 4 (Stumpf, P. K., ed.), pp. 321-362, Academic Press 6 Mackender, R. O. and Leech, R. M. (1974) Pl. Physiol. 53,496--502 7 Shipley, G. G., Green, J. P. and Nichols, B. W. (1973 ) Biochim. Biophys. Acta 311,531-544 8 Luzzati, V., Gulik-Krzywicki, T., Rivas, E., Reiss-Husson, F. and Rand, R. P. (1968)J. Gen. Physiol. 5 l, 37-43 9 Sen, A., Williams, W P. and Quinn, P. J. (1981) Biochim. Biophys. Acta 663,380-389 10 Gounaris, K., Mannock, D. A., Sen, A., Brain, A. P. R., Williams, W. P. and Quinn, P. J. (1983) Biochim. Biophys. Acta 732, 229-242 11 Bishop, D. G., Kenrick, J. R., Bayston, J. H., Macpherson, A. S. and Johns, S. R. (1980) Biochim. Biophys. Acta 602, 248-259 12 Quinn, P. J. and Williams, W. P. (1983) Biochim. Biophys. Acta 737, 223-266 13 Gounaris, K., Sen, A., Bmm, A. P. R., Quinn, P. J. and Williams, W. P. (1983) Biochim. Biophys• Acta 728, 129-139
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T1BS - October 1983 14 Gounaris, K., Brain, A. P. R., Quinn, P. J. and Williams, W. P. (1983)FEBS Lett. 153, 47-51 15 lsraelachvili, J. N., Marcelja, S. and Horn, R. G. (1980)Q. Rev. Biophys. 13, 121-200 16 Murphy, D. J. (1982)FEBS Lett. 150, 19-26 17 Barber, J. (1983) Plant, Cell and Environ. 6, 311-322 18 Gounaris, K., Sundby, C., Andersson, B. and
Barber, J. (1983)FEBSLett. 156, 171)-174 19 Anderson, J. M. and Andersson, B. (1982) Trends Biochem. Sci. 7,288-292 20 Uniu, M. D. and Hat'wood, J. L. (1982) in Developments in Plant Biology, Vol. 8 (Wintermans, J. F. G. M. and Kiiiper, P. J. C., eds.), pp. 359-362, Elsevier Biomedical Press 21 Sundby, C. and Larsson, C. 6th International
Congress in Photosynthesis (in press) 22 Siefemmnn-Harms, D., Ross, J. W., Kaneshiro, K. H. and Yamamoto, H. Y. (1982) FEBS Lett. 149, 191-196 23 Ford, R. C. and Barber, J. (1983) Biochim. BIOphys. Acta 722, 341-348 24 Chapman, D. J., DeFelice, J. G. and Barber, J. (1983) Plant Physiol. 72, 225-228
50 Years Ago then advances in this field have been enormous, mainly because of improved techniques and equipment. The work of Hans Fischer and his colleagues (and their Charles H. Gray contemporaries) was of the highest importFifty years ago, knowledge of the bile pig- jaundice, ethanol had to be added for the ance. Their techniques were those of clasments was limited to a few compounds coiour reaction to take place (the so-called sical organic chemistry - isolation, purifiindirect reaction); in obstructive jaundice cation, elemental analysis to provide an which had been isolated from natural sources; their chemical nature was eluci- the red colour developed without the addi- empirical formula, determination of dated by the methods of classical chemis- tion of ethanol (the direct reaction). Many molecular weight, derivation and degradablood specimens gave an intermediate reac- tion were all necessary preliminaries to try. Early in the nineteenth century, Tiedemann and Gmelin had shown that the tion and were presumed to contain both indicate the structure to be confh-med by synthesis. The system of organic microdirect and indirect bilirubin. yellow pigment in bile and serum and urine By the 1920s Kiister had recognized that analysis developed by Pregl was important of jaundiced subjects was dehydrogenated to green, blue, violet, red, orange and yel- the structure of bilirubin is closely related in this field. The monograph by Fischer and low pigments by nitric acid containing a to that of haem, the prosthetic group of Orth, published in 1937, 3 is a monument to trace of nitrous acid. These changes were haemoglobin; however, the structure of their contribution to the classical work in later found to be due to the formation from bilirubin was not finally elucidated until the bile pigment field. The bilene urobilin 1933, shortly after the structure of haem which has two ethyl groups instead of vinyl bilirubin of green biliverdins, blue and violet biliviolins, red bilirhodins and other itself had been established. Bilirubin was groups was synthesized in 1936 by Siedel coloured oxidation products. Before the shown to be a linear tetrapyrrole and haem a and MeieP, who were both pupils of Hans end of the nineteenth century, bilirubin had cyclic tetrapyrrole; the groups in the side Fischer. The synthesis of bilirubin (a bilidiene) was not accomplished until 1942 been isolated from gallstones and the chains were identical in nature and order2. The above is a brief account of what was by Fischer and PlieningeP. This was a fororange-red pigments urobilin and stercobilin had been found in pathological urine and known of the bile pigments in 1933. Since midable task because of the vinyl groups in two of the r-positions of the four pyrrole in faeces, respectively. Urobilinogen, a rings, and the asymmetry of its linear struccolourless precursor of urobilin in pathologture. ical urines, was shown in 1911 to be identiClassical separation techniques have cal to mesobilirubinogen formed by amalbeen replaced by column, paper, thin layer gam reduction of bilirubin. Biliverdin was and especially high performance high isolated from dog placentae by Lemberg pressure) liquid chromatography. The and Barcroft in 1932. crude polarimeters, colorimeters and specThe early view that bile pigment was troscopes have been superseded by formed in the liver was shown conclusively automated equipment which can record in a to be wrong1; bile pigments are formed in few minutes measurements which in 1933 the cells of the reticuloendothelial system would have taken a day or more to comand carried in the blood to the liver for plete, if they could have been carried out at excretion in the bile. In 1933, jaundice had all. Spectrophotometers for the ultraviolet long been recognized to be due to an and infrared wavelengths of radiation increased concentration of bilirubin in the became available after 1933 and such blood, either as a result of excessive equipment now can record the absorption haemolysis of red blood cells in the curve as a printout or even plot a series of reticuloendothelial system (retention jauncurves o n l i n e for the effluents obtained by dice) or obstruction in the biliary passages HPLC. Improved polarimetry, and mass by stone or tumour (obstructive jaundice). spectrometry, measurement of circular So-called catarrhal jaundice was believed to dichroism, X-ray diffraction and nuclear be caused by obstruction of the bile ducts by ~: magnetic resonance investigations have catarrh. Colorimetric tests for bile pigments revolutionized our knowledge of the in body fluids had been developed and the detailed structure of the bile pigments. diazo reaction was applied by van den Of major importance was the discovery Bergh to provide a quantitative estimation Reproduced by permission of P. A. Norstedt pubin 1943 by Dible, McMichael and Sherof bilirubin in plasma or serum. In retention lishers, Stockholm.
The bile pigments 1933-1983
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