- Email: [email protected]
[24] Membrane lipids
[24]
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The 37-kDa protein migrates at apparent molecular mass of 45 kDa in SDS-polyacrylamide gels, when plasma membranes are solubilized with SDS at 100°. The heat-induced band at 45 kDa on SDS-polyacrylamide gel electrophoresis is distinct from the one due to the above-described 42kDa protein. A corresponding heat-modifiable protein which cross-reacts with antibody against the 37-kDa protein ofA. nidulans is found in plasma membranes from Synechocystis PCC 671416 and Synechocystis PCC 6 8 0 3 . 9 The protein from Synechocystis PCC 6714 is found to bind carotenoids. 16 27 N. Murata, N. Sato, T. Omata, and T. Kuwabara, Plant Cell Physiol. 22, 855 (1981). 28 U. K. Laemmli, Nature (London) 227, 680 (1970).
[24] M e m b r a n e Lipids B y NAOKI SATO a n d NORIO M U R A T A
Introduction Cyanobacterial cells contain two types of membrane, the plasma membrane and thylakoid membranes, which are distinct from each other in their composition of proteins, lipids, and pigments. 1 The composition of the fatty acids of the lipids in both types of membrane changes with growth temperature so that cyanobacterial cells adapt themselves to the environmental temperature. 2,3 Major lipid classes in cyanobacterial membranes are monogalactosyl diacylglycerol (MGDG), monoglucosyl diacylglycerol (GIcDG), digalactosyl diacylglycerol (DGDG), sulfoquinovosyl diacylglycerol (SQDG), and phosphatidylglycerol (PG). 4,5 Fatty acids of cyanobacteria are mainly unbranched chains containing 14, 16, or 18 carbon atoms and 0, l, 2, or 3 double bonds. 4,6.7 Among them, palmitic acid and palmitoleic acid are most commonly found. The biosynthetic pathway of glycolipids in cyanobacteria is unique in that the glucolipid is a precursor to galactolipids ~.8 and stearic acid is esterified to lipids before 1 T. 2 N. 3 H. 4 N. 5 N. 6 C. 7 C. s T.
Omata and N. Murata, Plant Cell Physiol. 24, 1101 (1983). Sato and N. Murata, Biochim. Biophys. Acta 619, 353 (1980). Wada, R, Hirasawa, T. Omata, and N. Murata, Plant Cell Physiol. 25, 907 (1984). Sato, N. Murata, Y. Miura, and N. Ueta, Biochim. Biophys. Acta 572, 19 (1979). Sato and N. Murata, Biochim. Biophys. Acta 710, 271 (1982). N. Kenyon, J. Bacteriol. 109, 827 (1972). N. Kenyon, R. Rippka, and R. Y. Stanier, Arch. Mikrobiol. 83, 216 (1972). Omata and N. Murata, Plant Cell Physiol. 27, 485 (1986).
METHODS IN ENZYMOLOGY, VOL. 167
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being desaturated to oleic acid. 9:° This chapter describesprocedures in cyanobacterial lipid analysis, including extraction and fractionation of lipids, analysis of their fatty acids, determination of positional distribution of fatty acids within the lipids, and analysis of lipid molecular species. The interested reader should consult a textbook tl for general experimental procedures in lipid analysis. Analytical data of cyanobacterial iipids and fatty acids are available.l.2,4-9.12 Extraction of Total Lipids Total lipids are extracted from either intact cells or membrane preparations of cyanobacteria according to the method of Bligh and Dyer) 3 When working with lipids that contain highly unsaturated fatty acids (such as those from most filamentous cyanobacteria), excessive exposure of unsolvated lipids to air should be minimized to prevent fatty acid oxidation. The presence of an antioxidant [e.g., 2,6-di-tert-butyl-4-hydroxytoluene (BHT)] is recommended during long-term storage of lipids containing unsaturated fatty acids. Procedure. One-tenth milliliter of pelleted cyanobacterial cells or membranes is suspended in 1 ml of distilled water or dilute buffer (high concentrations of sugars and other osmotics should be washed out beforehand). The suspension is transferred to a glass tube with a Teflon-lined screw-cap (e.g., Pyrex No. 8082CTF centrifuge tube). Then 3.75 ml of CHCI3/CHaOH (1 : 2, v/v) is added and mixed by vortexing. After standing for 20 min at room temperature, 1.25 ml each of CHC13 and H20 are added to the mixture and mixed by vortexing. The mixture is then centrifuged at 1,000 g for 15 min at room temperature. The clear upper phase and the intermediate fluff layer are carefully withdrawn with a Pasteur pipet and discarded. Then 2.5 ml of CH3OH/H20 (10 : 9) is added to the lower phase and mixed by vortexing, after which the mixture is centrifuged as above. The lower phase is recovered and transferred to a new test tube suitable for evaporation. One-half milliliter of C2H5OH is added for complete evaporation of water, and the solvent is evaporated under reduced pressure using a rotary evaporator or under a stream of N2. The total lipids are dissolved in 0.5 ml of CHCI3/CH3OH (2 : 1, v/v). For storage times up to a few hours, the tube is covered with aluminum foil and chilled on ice to 9 N. 10 N. 11 M. 12 N. 13 E.
Sato and N. Murata, Biochim. Biophys. Acta 710, 279 (1982). W. Lem and P. K. Stumpf, Plant Physiol. 74, 134 (1984). Kates, "Techniques of Lipidology." North-Holland, Amsterdam, 1972. Murata and N. Sato, Plant Cell Physiol. 2,4, 133 (1983). G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).
[24]
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minimize evaporation of the solvent. For longer storage times, BHT is added to a final concentration of 0.05%, then the lipid solution is transferred to a small glass vial with a Teflon-lined screw-cap and stored below - 2 0 °. BHT can be removed by thin-layer chromatography. Thin-Layer Chromatographic Separation of Lipid Classes Thin-layer chromatography (TLC) is a convenient method for separating major classes of lipids. Minor amounts of unidentified lipids may also be present, but they usually do not interfere with the analysis of the major classes of lipids, since they remain near the origin in the TLC systems described below. Procedure. A pencil line is drawn on a precoated silica gel plate (e.g., Merck No. 5721 plate, without fluorescent dye, 5 x 20 cm or 20 × 20 cm) parallel to and approximately 2 cm from one edge. Lipid solution (10-50 /zl) is applied as a streak approximately 2 cm long along the pencil line, with a microsyringe. The solvent may be removed more rapidly with an air blower. The plate is developed with either (1) (CH3)2CO/C6H6/H20 (91 : 30 : 8, by volume) or (2) CHCI3/CH3OH/NH4OH (28%) (13 : 7 : 1, by volume) to a height of 10-19 cm. Solvent (2) is suitable for separating MGDG, PG, SQDG, and DGDG, and Solvent (1) separates MGDG and GIcDG (Fig. 1). The plate is allowed to dry in a fume hood for about 20 min, then sprayed with 0.01% primuline in (CH3)2CO/H20 (4:1). The plate is again allowed to dry for 3 min. Lipids are detected under longwavelength UV light (366 nm) as bluish white fluorescent bands, which are outlined with a pencil. When the lipids are recovered from the plate, the silica gel within outlined areas is scraped into a flask and extracted in CHC13/CH3OH (2 : 1). The mixture is filtered through a sheet of filter paper. Water-soluble impurities may be removed by washing the filtrate with ¼ volume of distilled water. Then the solution is concentrated as described above under Extraction of Total Lipids. Comments. Typical separation patterns are illustrated in Fig. 1. Lipid classes are easily identified by their mobility on TLC. Spray reagents are conveniently used for identification: anthrone reagent for glycolipids and Dittmer reagent for phospholipids are especially useful (see Ref. I 1 for the preparation and use of spray reagents). Definitive identification can only be effected by infrared and nuclear magnetic resonance spectrometry (see a textbook 11for a general introduction). MGDG and GlcDG can be distinguished by gas chromatographic analysis of their sugar moieties. 5 Since the content of GIcDG is much lower than that of the other four major classes of lipids, 5,12 analysis of GlcDG requires a larger scale, e.g.,
254
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND A
N2 FIXATION
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-~ Front
B -~Front )1
74
~5 ~6
~Origin FIG. 1. Thin-layer chromatograms of total lipids from Anabaena variabilis strain M3. (A) A Merck 5721 plate was developed in Solvent (1) to a height of 18 cm. (B) A Merck 5721 plate was developed in Solvent (2) to a height of 10 cm. A 10-cm plate is sufficient to separate MGDG, DGDG, SQDG, and PG in Solvent (2), whereas good separation of GIcDG and MGDG requires a longer plate using Solvent (1). Identification: 1, pigments; 2, monoglucosyl diacylglycerol (GIcDG); 3, monogalactosyl diacylglycerol (MGDG); 4, digalactosyl diacylglycerol (DGDG); 5, sulfoquinovosyl diacylglycerol (SQDG); 6, phosphatidylglycerol (PG).
about 1 liter of culture in middle exponential growth (-5/.d packed cells/ ml). In this case, the lipid extraction and TLC should be scaled up 10-fold. If the separation of GIcDG from MGDG is incomplete, then the GIcDG fraction should be recovered and repurified by TLC. Gas
Chromatographic Analysis of Fatty Acids in Lipids
The content and composition of fatty acids in lipids are determined by gas chromatographic analysis of the methyl esters which arc obtained by methanolysis of the lipids. Methanolysis is generally performed without isolating lipids from the silica gel. Fatty acid methyl esters of cyanobacteria are easily identified in gas chromatographic analysis, though definitive identification and determination of double bond position are effected by
[24]
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gas chromatography-mass spectrometry (GC-MS). Consult the literature for technical details of gas chromatography. 14 Procedure. After the detection of lipids by primuline fluorescence, the TLC plate is dried in a vacuum desiccator for 30 rain. The silica gel in the appropriate area is scraped with a razor blade into a test tube (15 mm diameter by 15 cm length) with a Teflon-lined screw-cap (e.g., Pyrex TSTSCR 16-150 tube). Three milliliters of 2.5% (w/w) HCI in anhydrous CH3OH (for this solution, HCI gas is bubbled into the CH3OH until its weight increases by 2.5%) and 100/zl of 1 mM pentadecanoic acid as an internal standard in benzene are added. The tube is tightly capped and heated at 85 ° for 2.5 hr in an aluminum heating block or in a water bath. After the tube is cooled to room temperature, 2.5 ml of n-hexane is added and mixed by vortexing. After standing for 5 min, the upper (hexane) phase is carefully transferred to a clean test tube with a narrowly tapered bottom suitable for evaporation. The lower phase is extracted twice more with additional hexane. Then 2 ml of distilled water is added to the remaining lower phase, and hexane extraction is repeated once more. After combining the hexane extracts, the solvent is removed by evaporation under reduced pressure or a stream of N2. The resulting methyl esters are dissolved in 20 /zl of n-hexane. The tube is covered with a piece of aluminum foil (avoid using paraffin film) and placed on ice until analysis. A 5-/xl portion of the methyl ester solution is injected into a gas chromatograph equipped with a glass column (3 mm × 2 m) packed with 15% diethylene glycol succinate on Chromosorb W (acid washed). Temperatures of the column and the flame ionization detector are 180 and 260° , respectively. The flow rate of the N2 or He gas is 30 ml/min. A chromatography data processor (e.g., Chromatopac R3A, Shimadzu Seisakusho, Kyoto) is used to record and store chromatograms and to calculate peak areas. Comments. Figure 2 shows a typical gas chromatogram of fatty acid methyl esters of MGDG from Anabaena variabilis. By using appropriate calibration factors (e.g., assuming that the ratio of integrated areas is identical to the mass ratio of methyl esters of fatty acids), both fatty acid composition and the total amount of fatty acids can be calculated. Then the total amount of each lipid class can be calculated by dividing the amount of fatty acids by 2. Positional Distribution of Fatty Acids in Lipids The position-specific hydrolysis of lipids by lipase is used to analyze the fatty acids esterified to the sn-I and sn-2 positions of the glycerol 14R. G. Ackman, this series, Vol. 14, p. 329.
256
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND N 2 FIXATION
[24]
1t
c O
2
t
0
5 Time,
~
~
8
I
i
10 rain
15
FIG. 2. Gas chromatogram of fatty acid methyl esters prepared from monogalactosyl diacylglycerol inAnabaena variabilis strain M3. Identification: 1, pentadecanoic acid (15 : 0, internal standard); 2, palmitic acid (16 : 0); 3, palmitoleic acid (16 : 1); 4, hexadecadienoic acid (16 : 2); 5, stearic acid (18 : 0); 6, oleic acid (18 : 1); 7, linoleic acid (18 : 2); 8, t~-linolenic acid (18 : 3).
moiety. The lipase from Rhizopus delemar, which liberates fatty acids at the sn-1 position of glycolipids and phospholipids, is suitable for this purpose. 2,9A5 Procedure. Between 0.1 and I mg of lipid, purified by TLC as described above for Thin-Layer Chromatographic Separation of Lipid Classes and dissolved in CHC13/CH3OH (2: 1) is taken to dryness under reduced pressure or under a stream of N2, then suspended in 0.9 ml of 50 mM tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 7.2), 0.05% Triton X-100, and mixed by vortexing. About 0.5 mg of Rhizopus lipase (6000 U/mg, pure grade, Seikagaku Kogyo, Tokyo), which is suspended in 0. I ml of the same buffer, is added and mixed. The mixture is incubated at 37° for 10-60 min with gentle shaking (the reaction time should be controlled according to lipid classes as described below in Comments). Then 3 ml of C2HsOH is added to the mixture, and the solvent is removed using a rotary evaporator. Water should be removed completely. One-tenth milliliter of CHCI3/CH3OH (2 : 1) is added to the residue and swirled gently in an ice-water bath (the low temperature prevents solubilization of Tris). The supernatant is applied to a TLC plate (5 × 20 cm), which is developed in Solvent (1) (see above) for GIcDG and MGDG or in CHC13/ t5 W. Fischer, E. Heinz, and M. Zeus, Hoppe-Seyler's Z. Physiol. Chem. 354, 1115 (1973).
[24]
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257
(CH3)2CO/CH3OH/CH3COOH/H20 ( 1 0 : 4 : 2 : 3 : 1, by volume) for DGDG, SQDG, and PG. Reference compounds (purified lipids and free fatty acids) should be developed on the same plate for identification of the lysolipids and free fatty acids. Lysolipids and free fatty acids are located by primuline fluorescence as described above. Free fatty acids run just below the solvent front, whereas lysolipids appear below the original diacyl lipids. Note that Triton X-100 appears as a broad band near the solvent front. The silica gel in the fatty acid and lysolipid bands is scraped and used for fatty acid analysis as described above for Gas Chromatographic Analysis of Fatty Acids in Lipids. Comments. The reaction time should be controlled so that the hydrolysis is just completed. Longer incubation results in partial hydrolysis of the lysolipid. Under the conditions given above, GIcDG and MGDG are hydrolyzed within 10 min, while the hydrolysis of DGDG, SQDG, and PG requires longer times ( - 2 0 min for DGDG, 30-60 min for SQDG and PG). The fatty acid composition at the sn-2 position is determined by analyzing the fatty acids in the lysolipid. The fatty acid composition at the sn-1 position may be either determined directly from analysis of free fatty acids or estimated by comparing the fatty acid compositions of the original diacyl lipid and the lysolipid. The latter method is preferable since the free fatty acids which are located near the solvent front on the TLC plate are often contaminated. Molecular Species of Lipids Each class of lipids is a mixture of molecular species which contain various combinations of fatty acids at the sn-I and sn-2 positions. Molecular species of glycolipids are separated by TLC using a AgNO3-impregnated silica gel plate. The information on the positional distribution of fatty acids is necessary for the complete determination of the composition of molecular species. Procedure. A Merck 5721 plate (10 x 20 cm) is immersed for 20 min in 5% (w/v) AgNO3 in CH3CN poured in a clean enamel dish of the type normally used for photography processing. The AgNO3 solution can be stored in an amber bottle at 4 ° and used repeatedly. Gloves should be worn during manipulation. The plate is dried in a fume hood and activated at 105° for 30 min. The time and temperature of activation should be controlled so that the plate is not darkened. Purified lipid (see Thin-Layer Chromatographic Separation of Lipid Classes) dissolved in CHCI3/CH3OH (2 : 1) is applied to a AgNO3-impregnated plate, and the plate is developed in Solvent (1) (see above) for GIcDG and MGDG and in CHC13/CH3OH/H~O (60 : 30 : 4, by volume) for
258
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND
N2 FIXATION
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Front
sn-1/sn-2 18:0/16:0 18:1/16:0 16:2/16:0 18:1/16:1 18:2/16:1 18:3/16:0 18:3/16:1
~-Origin
FIG. 3. Thin-layer chromatogram of molecular species of monogalactosyl diacylglycerol (MGDG) from Anabaena variabilis. Identification of molecular species is shown by combination of abbreviated forms of fatty acids (see the legend to Fig. 2 for names of fatty acids corresponding to specific number designations).
DGDG and SQDG to a height of 19 cm. Molecular species are detected by primuline fluorescence as described above. The use of old plates or excessively activated plates gives high background fluorescence and makes it difficult to detect lipid bands. Each lipid molecular species is extracted from the silica gel in 4 ml of CHCI3/CH3OH (2: 1). Silica gel is removed by centrifugation at 1,000 g for 10 min. The supernatant is transferred to another tube, One milliliter of H20 is added and mixed by vortexing. After centrifugation as above the upper layer is removed and discarded. The lower layer is washed twice with 1 ml of H20. These washing steps are necessary to remove AgNO3 which would otherwise oxidize unsaturated fatty acids during methanolysis. The lipid molecular species are mixed with an internal standard (pentadecanoic acid) and subjected to methanolysis as described above for gas chromatography of fatty acids. Based on the positional distribution of fatty acids (see above) and fatty acid composition determined here, each molecular species of lipids is identified. The amount of molecular species is determined from the content of fatty acids, and then the composition of lipid molecular species is calculated. Comments. A typical separation of molecular species of MGDG from Anabaena variabilis is shown in Fig. 3. Each molecular species is identified by its fatty acid composition. The separation is mainly effected ac-
RAPID PERMEABILIZATIONOF A. nidulans
[25]
259
cording to the number of double bonds in the molecular species. However, 18 : 1/16 : 1 and 18 : 2/16 : 0, as well as 18:2/16 : 1 and 18 : 3/16 : 0, can be separated as distinct bands in close proximity. Molecular species of DGDG and SQDG are also analyzed by this technique, but their separation is inferior to that of molecular species of GIcDG and MGDG. Molecular species of PG can be analyzed as acetyldiacylglycerol, which is prepared by digestion of PG by phospholipase C and acetylation of the resulting diacylglycerol. 9,~6 16 M. Kito, M. Ishinaga, M. Nishihara, M. Kato, S. Sawada, and T. Hata, Eur. J. Biochem. 54, 55 (1975).
[25] R a p i d P e r m e a b i l i z a t i o n o f Anacystis nidulans to E l e c t r o l y t e s
By GEORGE C. PAPAGEORGIOU Introduction The cell envelope of gram-negative bacteria (studied mainly in enteric bacteria) 1 consists of two hydrophobic layers, the outer membrane and the cell membrane, and a hydrophilic space, the periplasm, sandwiched in between. Peptidoglycan, an open network of polysaccharide backbones (alternating N-acetylglucosamine and N-acetylmuramic acid residues) cross-linked with oligopeptides and covalently linked to outer membrane proteins, occupies the periplasmic space next to the cell membrane. It serves as the cell exoskeleton. The cell membrane is the true selective permeability barrier. The outer membrane is only a passive molecular sieve. In gram-negative cyanobacteria intact peptidoglycan is essential for cell envelope impermeability. Partial or total enzymic hydrolysis of peptidoglycan with lysozyme yields cells permeable to ions. 2-4 Although other means of permeabilization exist, 5,6 the enzymic method is superior when preservation of photosynthetic activity is desired. Different I H. Nikaido and M. Vaara, Microbiol. Rev. 49, 1 (1985). 2 j. Biggins, Plant Physiol. 42, 1442 (1967). 3 B. Ward and J. Myers, Plant Physiol. 50, 547 (1972). 4 G. C. Papageorgiou and T. Lagoyanni, Biochim. Biophys. Acta 807, 230 (1985). 5 B. Gerhardt and A. Trebst, Z. Naturforsch. B 20, 879 (1965). 6 S. J. Robinson, C. S. DeRoo, and C. F. Yocum, Plant Physiol. 70, 154 (1982).
METHODS IN ENZYMOLOGY. VOL. 167
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The 37-kDa protein migrates at apparent molecular mass of 45 kDa in SDS-polyacrylamide gels, when plasma membranes are solubilized with SDS at 100°. The heat-induced band at 45 kDa on SDS-polyacrylamide gel electrophoresis is distinct from the one due to the above-described 42kDa protein. A corresponding heat-modifiable protein which cross-reacts with antibody against the 37-kDa protein ofA. nidulans is found in plasma membranes from Synechocystis PCC 671416 and Synechocystis PCC 6 8 0 3 . 9 The protein from Synechocystis PCC 6714 is found to bind carotenoids. 16 27 N. Murata, N. Sato, T. Omata, and T. Kuwabara, Plant Cell Physiol. 22, 855 (1981). 28 U. K. Laemmli, Nature (London) 227, 680 (1970).
[24] M e m b r a n e Lipids B y NAOKI SATO a n d NORIO M U R A T A
Introduction Cyanobacterial cells contain two types of membrane, the plasma membrane and thylakoid membranes, which are distinct from each other in their composition of proteins, lipids, and pigments. 1 The composition of the fatty acids of the lipids in both types of membrane changes with growth temperature so that cyanobacterial cells adapt themselves to the environmental temperature. 2,3 Major lipid classes in cyanobacterial membranes are monogalactosyl diacylglycerol (MGDG), monoglucosyl diacylglycerol (GIcDG), digalactosyl diacylglycerol (DGDG), sulfoquinovosyl diacylglycerol (SQDG), and phosphatidylglycerol (PG). 4,5 Fatty acids of cyanobacteria are mainly unbranched chains containing 14, 16, or 18 carbon atoms and 0, l, 2, or 3 double bonds. 4,6.7 Among them, palmitic acid and palmitoleic acid are most commonly found. The biosynthetic pathway of glycolipids in cyanobacteria is unique in that the glucolipid is a precursor to galactolipids ~.8 and stearic acid is esterified to lipids before 1 T. 2 N. 3 H. 4 N. 5 N. 6 C. 7 C. s T.
Omata and N. Murata, Plant Cell Physiol. 24, 1101 (1983). Sato and N. Murata, Biochim. Biophys. Acta 619, 353 (1980). Wada, R, Hirasawa, T. Omata, and N. Murata, Plant Cell Physiol. 25, 907 (1984). Sato, N. Murata, Y. Miura, and N. Ueta, Biochim. Biophys. Acta 572, 19 (1979). Sato and N. Murata, Biochim. Biophys. Acta 710, 271 (1982). N. Kenyon, J. Bacteriol. 109, 827 (1972). N. Kenyon, R. Rippka, and R. Y. Stanier, Arch. Mikrobiol. 83, 216 (1972). Omata and N. Murata, Plant Cell Physiol. 27, 485 (1986).
METHODS IN ENZYMOLOGY, VOL. 167
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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being desaturated to oleic acid. 9:° This chapter describesprocedures in cyanobacterial lipid analysis, including extraction and fractionation of lipids, analysis of their fatty acids, determination of positional distribution of fatty acids within the lipids, and analysis of lipid molecular species. The interested reader should consult a textbook tl for general experimental procedures in lipid analysis. Analytical data of cyanobacterial iipids and fatty acids are available.l.2,4-9.12 Extraction of Total Lipids Total lipids are extracted from either intact cells or membrane preparations of cyanobacteria according to the method of Bligh and Dyer) 3 When working with lipids that contain highly unsaturated fatty acids (such as those from most filamentous cyanobacteria), excessive exposure of unsolvated lipids to air should be minimized to prevent fatty acid oxidation. The presence of an antioxidant [e.g., 2,6-di-tert-butyl-4-hydroxytoluene (BHT)] is recommended during long-term storage of lipids containing unsaturated fatty acids. Procedure. One-tenth milliliter of pelleted cyanobacterial cells or membranes is suspended in 1 ml of distilled water or dilute buffer (high concentrations of sugars and other osmotics should be washed out beforehand). The suspension is transferred to a glass tube with a Teflon-lined screw-cap (e.g., Pyrex No. 8082CTF centrifuge tube). Then 3.75 ml of CHCI3/CHaOH (1 : 2, v/v) is added and mixed by vortexing. After standing for 20 min at room temperature, 1.25 ml each of CHC13 and H20 are added to the mixture and mixed by vortexing. The mixture is then centrifuged at 1,000 g for 15 min at room temperature. The clear upper phase and the intermediate fluff layer are carefully withdrawn with a Pasteur pipet and discarded. Then 2.5 ml of CH3OH/H20 (10 : 9) is added to the lower phase and mixed by vortexing, after which the mixture is centrifuged as above. The lower phase is recovered and transferred to a new test tube suitable for evaporation. One-half milliliter of C2H5OH is added for complete evaporation of water, and the solvent is evaporated under reduced pressure using a rotary evaporator or under a stream of N2. The total lipids are dissolved in 0.5 ml of CHCI3/CH3OH (2 : 1, v/v). For storage times up to a few hours, the tube is covered with aluminum foil and chilled on ice to 9 N. 10 N. 11 M. 12 N. 13 E.
Sato and N. Murata, Biochim. Biophys. Acta 710, 279 (1982). W. Lem and P. K. Stumpf, Plant Physiol. 74, 134 (1984). Kates, "Techniques of Lipidology." North-Holland, Amsterdam, 1972. Murata and N. Sato, Plant Cell Physiol. 2,4, 133 (1983). G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).
[24]
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minimize evaporation of the solvent. For longer storage times, BHT is added to a final concentration of 0.05%, then the lipid solution is transferred to a small glass vial with a Teflon-lined screw-cap and stored below - 2 0 °. BHT can be removed by thin-layer chromatography. Thin-Layer Chromatographic Separation of Lipid Classes Thin-layer chromatography (TLC) is a convenient method for separating major classes of lipids. Minor amounts of unidentified lipids may also be present, but they usually do not interfere with the analysis of the major classes of lipids, since they remain near the origin in the TLC systems described below. Procedure. A pencil line is drawn on a precoated silica gel plate (e.g., Merck No. 5721 plate, without fluorescent dye, 5 x 20 cm or 20 × 20 cm) parallel to and approximately 2 cm from one edge. Lipid solution (10-50 /zl) is applied as a streak approximately 2 cm long along the pencil line, with a microsyringe. The solvent may be removed more rapidly with an air blower. The plate is developed with either (1) (CH3)2CO/C6H6/H20 (91 : 30 : 8, by volume) or (2) CHCI3/CH3OH/NH4OH (28%) (13 : 7 : 1, by volume) to a height of 10-19 cm. Solvent (2) is suitable for separating MGDG, PG, SQDG, and DGDG, and Solvent (1) separates MGDG and GIcDG (Fig. 1). The plate is allowed to dry in a fume hood for about 20 min, then sprayed with 0.01% primuline in (CH3)2CO/H20 (4:1). The plate is again allowed to dry for 3 min. Lipids are detected under longwavelength UV light (366 nm) as bluish white fluorescent bands, which are outlined with a pencil. When the lipids are recovered from the plate, the silica gel within outlined areas is scraped into a flask and extracted in CHC13/CH3OH (2 : 1). The mixture is filtered through a sheet of filter paper. Water-soluble impurities may be removed by washing the filtrate with ¼ volume of distilled water. Then the solution is concentrated as described above under Extraction of Total Lipids. Comments. Typical separation patterns are illustrated in Fig. 1. Lipid classes are easily identified by their mobility on TLC. Spray reagents are conveniently used for identification: anthrone reagent for glycolipids and Dittmer reagent for phospholipids are especially useful (see Ref. I 1 for the preparation and use of spray reagents). Definitive identification can only be effected by infrared and nuclear magnetic resonance spectrometry (see a textbook 11for a general introduction). MGDG and GlcDG can be distinguished by gas chromatographic analysis of their sugar moieties. 5 Since the content of GIcDG is much lower than that of the other four major classes of lipids, 5,12 analysis of GlcDG requires a larger scale, e.g.,
254
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND A
N2 FIXATION
[24]
-~ Front
B -~Front )1
74
~5 ~6
~Origin FIG. 1. Thin-layer chromatograms of total lipids from Anabaena variabilis strain M3. (A) A Merck 5721 plate was developed in Solvent (1) to a height of 18 cm. (B) A Merck 5721 plate was developed in Solvent (2) to a height of 10 cm. A 10-cm plate is sufficient to separate MGDG, DGDG, SQDG, and PG in Solvent (2), whereas good separation of GIcDG and MGDG requires a longer plate using Solvent (1). Identification: 1, pigments; 2, monoglucosyl diacylglycerol (GIcDG); 3, monogalactosyl diacylglycerol (MGDG); 4, digalactosyl diacylglycerol (DGDG); 5, sulfoquinovosyl diacylglycerol (SQDG); 6, phosphatidylglycerol (PG).
about 1 liter of culture in middle exponential growth (-5/.d packed cells/ ml). In this case, the lipid extraction and TLC should be scaled up 10-fold. If the separation of GIcDG from MGDG is incomplete, then the GIcDG fraction should be recovered and repurified by TLC. Gas
Chromatographic Analysis of Fatty Acids in Lipids
The content and composition of fatty acids in lipids are determined by gas chromatographic analysis of the methyl esters which arc obtained by methanolysis of the lipids. Methanolysis is generally performed without isolating lipids from the silica gel. Fatty acid methyl esters of cyanobacteria are easily identified in gas chromatographic analysis, though definitive identification and determination of double bond position are effected by
[24]
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gas chromatography-mass spectrometry (GC-MS). Consult the literature for technical details of gas chromatography. 14 Procedure. After the detection of lipids by primuline fluorescence, the TLC plate is dried in a vacuum desiccator for 30 rain. The silica gel in the appropriate area is scraped with a razor blade into a test tube (15 mm diameter by 15 cm length) with a Teflon-lined screw-cap (e.g., Pyrex TSTSCR 16-150 tube). Three milliliters of 2.5% (w/w) HCI in anhydrous CH3OH (for this solution, HCI gas is bubbled into the CH3OH until its weight increases by 2.5%) and 100/zl of 1 mM pentadecanoic acid as an internal standard in benzene are added. The tube is tightly capped and heated at 85 ° for 2.5 hr in an aluminum heating block or in a water bath. After the tube is cooled to room temperature, 2.5 ml of n-hexane is added and mixed by vortexing. After standing for 5 min, the upper (hexane) phase is carefully transferred to a clean test tube with a narrowly tapered bottom suitable for evaporation. The lower phase is extracted twice more with additional hexane. Then 2 ml of distilled water is added to the remaining lower phase, and hexane extraction is repeated once more. After combining the hexane extracts, the solvent is removed by evaporation under reduced pressure or a stream of N2. The resulting methyl esters are dissolved in 20 /zl of n-hexane. The tube is covered with a piece of aluminum foil (avoid using paraffin film) and placed on ice until analysis. A 5-/xl portion of the methyl ester solution is injected into a gas chromatograph equipped with a glass column (3 mm × 2 m) packed with 15% diethylene glycol succinate on Chromosorb W (acid washed). Temperatures of the column and the flame ionization detector are 180 and 260° , respectively. The flow rate of the N2 or He gas is 30 ml/min. A chromatography data processor (e.g., Chromatopac R3A, Shimadzu Seisakusho, Kyoto) is used to record and store chromatograms and to calculate peak areas. Comments. Figure 2 shows a typical gas chromatogram of fatty acid methyl esters of MGDG from Anabaena variabilis. By using appropriate calibration factors (e.g., assuming that the ratio of integrated areas is identical to the mass ratio of methyl esters of fatty acids), both fatty acid composition and the total amount of fatty acids can be calculated. Then the total amount of each lipid class can be calculated by dividing the amount of fatty acids by 2. Positional Distribution of Fatty Acids in Lipids The position-specific hydrolysis of lipids by lipase is used to analyze the fatty acids esterified to the sn-I and sn-2 positions of the glycerol 14R. G. Ackman, this series, Vol. 14, p. 329.
256
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND N 2 FIXATION
[24]
1t
c O
2
t
0
5 Time,
~
~
8
I
i
10 rain
15
FIG. 2. Gas chromatogram of fatty acid methyl esters prepared from monogalactosyl diacylglycerol inAnabaena variabilis strain M3. Identification: 1, pentadecanoic acid (15 : 0, internal standard); 2, palmitic acid (16 : 0); 3, palmitoleic acid (16 : 1); 4, hexadecadienoic acid (16 : 2); 5, stearic acid (18 : 0); 6, oleic acid (18 : 1); 7, linoleic acid (18 : 2); 8, t~-linolenic acid (18 : 3).
moiety. The lipase from Rhizopus delemar, which liberates fatty acids at the sn-1 position of glycolipids and phospholipids, is suitable for this purpose. 2,9A5 Procedure. Between 0.1 and I mg of lipid, purified by TLC as described above for Thin-Layer Chromatographic Separation of Lipid Classes and dissolved in CHC13/CH3OH (2: 1) is taken to dryness under reduced pressure or under a stream of N2, then suspended in 0.9 ml of 50 mM tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 7.2), 0.05% Triton X-100, and mixed by vortexing. About 0.5 mg of Rhizopus lipase (6000 U/mg, pure grade, Seikagaku Kogyo, Tokyo), which is suspended in 0. I ml of the same buffer, is added and mixed. The mixture is incubated at 37° for 10-60 min with gentle shaking (the reaction time should be controlled according to lipid classes as described below in Comments). Then 3 ml of C2HsOH is added to the mixture, and the solvent is removed using a rotary evaporator. Water should be removed completely. One-tenth milliliter of CHCI3/CH3OH (2 : 1) is added to the residue and swirled gently in an ice-water bath (the low temperature prevents solubilization of Tris). The supernatant is applied to a TLC plate (5 × 20 cm), which is developed in Solvent (1) (see above) for GIcDG and MGDG or in CHC13/ t5 W. Fischer, E. Heinz, and M. Zeus, Hoppe-Seyler's Z. Physiol. Chem. 354, 1115 (1973).
[24]
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(CH3)2CO/CH3OH/CH3COOH/H20 ( 1 0 : 4 : 2 : 3 : 1, by volume) for DGDG, SQDG, and PG. Reference compounds (purified lipids and free fatty acids) should be developed on the same plate for identification of the lysolipids and free fatty acids. Lysolipids and free fatty acids are located by primuline fluorescence as described above. Free fatty acids run just below the solvent front, whereas lysolipids appear below the original diacyl lipids. Note that Triton X-100 appears as a broad band near the solvent front. The silica gel in the fatty acid and lysolipid bands is scraped and used for fatty acid analysis as described above for Gas Chromatographic Analysis of Fatty Acids in Lipids. Comments. The reaction time should be controlled so that the hydrolysis is just completed. Longer incubation results in partial hydrolysis of the lysolipid. Under the conditions given above, GIcDG and MGDG are hydrolyzed within 10 min, while the hydrolysis of DGDG, SQDG, and PG requires longer times ( - 2 0 min for DGDG, 30-60 min for SQDG and PG). The fatty acid composition at the sn-2 position is determined by analyzing the fatty acids in the lysolipid. The fatty acid composition at the sn-1 position may be either determined directly from analysis of free fatty acids or estimated by comparing the fatty acid compositions of the original diacyl lipid and the lysolipid. The latter method is preferable since the free fatty acids which are located near the solvent front on the TLC plate are often contaminated. Molecular Species of Lipids Each class of lipids is a mixture of molecular species which contain various combinations of fatty acids at the sn-I and sn-2 positions. Molecular species of glycolipids are separated by TLC using a AgNO3-impregnated silica gel plate. The information on the positional distribution of fatty acids is necessary for the complete determination of the composition of molecular species. Procedure. A Merck 5721 plate (10 x 20 cm) is immersed for 20 min in 5% (w/v) AgNO3 in CH3CN poured in a clean enamel dish of the type normally used for photography processing. The AgNO3 solution can be stored in an amber bottle at 4 ° and used repeatedly. Gloves should be worn during manipulation. The plate is dried in a fume hood and activated at 105° for 30 min. The time and temperature of activation should be controlled so that the plate is not darkened. Purified lipid (see Thin-Layer Chromatographic Separation of Lipid Classes) dissolved in CHCI3/CH3OH (2 : 1) is applied to a AgNO3-impregnated plate, and the plate is developed in Solvent (1) (see above) for GIcDG and MGDG and in CHC13/CH3OH/H~O (60 : 30 : 4, by volume) for
258
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND
N2 FIXATION
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Front
sn-1/sn-2 18:0/16:0 18:1/16:0 16:2/16:0 18:1/16:1 18:2/16:1 18:3/16:0 18:3/16:1
~-Origin
FIG. 3. Thin-layer chromatogram of molecular species of monogalactosyl diacylglycerol (MGDG) from Anabaena variabilis. Identification of molecular species is shown by combination of abbreviated forms of fatty acids (see the legend to Fig. 2 for names of fatty acids corresponding to specific number designations).
DGDG and SQDG to a height of 19 cm. Molecular species are detected by primuline fluorescence as described above. The use of old plates or excessively activated plates gives high background fluorescence and makes it difficult to detect lipid bands. Each lipid molecular species is extracted from the silica gel in 4 ml of CHCI3/CH3OH (2: 1). Silica gel is removed by centrifugation at 1,000 g for 10 min. The supernatant is transferred to another tube, One milliliter of H20 is added and mixed by vortexing. After centrifugation as above the upper layer is removed and discarded. The lower layer is washed twice with 1 ml of H20. These washing steps are necessary to remove AgNO3 which would otherwise oxidize unsaturated fatty acids during methanolysis. The lipid molecular species are mixed with an internal standard (pentadecanoic acid) and subjected to methanolysis as described above for gas chromatography of fatty acids. Based on the positional distribution of fatty acids (see above) and fatty acid composition determined here, each molecular species of lipids is identified. The amount of molecular species is determined from the content of fatty acids, and then the composition of lipid molecular species is calculated. Comments. A typical separation of molecular species of MGDG from Anabaena variabilis is shown in Fig. 3. Each molecular species is identified by its fatty acid composition. The separation is mainly effected ac-
RAPID PERMEABILIZATIONOF A. nidulans
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259
cording to the number of double bonds in the molecular species. However, 18 : 1/16 : 1 and 18 : 2/16 : 0, as well as 18:2/16 : 1 and 18 : 3/16 : 0, can be separated as distinct bands in close proximity. Molecular species of DGDG and SQDG are also analyzed by this technique, but their separation is inferior to that of molecular species of GIcDG and MGDG. Molecular species of PG can be analyzed as acetyldiacylglycerol, which is prepared by digestion of PG by phospholipase C and acetylation of the resulting diacylglycerol. 9,~6 16 M. Kito, M. Ishinaga, M. Nishihara, M. Kato, S. Sawada, and T. Hata, Eur. J. Biochem. 54, 55 (1975).
[25] R a p i d P e r m e a b i l i z a t i o n o f Anacystis nidulans to E l e c t r o l y t e s
By GEORGE C. PAPAGEORGIOU Introduction The cell envelope of gram-negative bacteria (studied mainly in enteric bacteria) 1 consists of two hydrophobic layers, the outer membrane and the cell membrane, and a hydrophilic space, the periplasm, sandwiched in between. Peptidoglycan, an open network of polysaccharide backbones (alternating N-acetylglucosamine and N-acetylmuramic acid residues) cross-linked with oligopeptides and covalently linked to outer membrane proteins, occupies the periplasmic space next to the cell membrane. It serves as the cell exoskeleton. The cell membrane is the true selective permeability barrier. The outer membrane is only a passive molecular sieve. In gram-negative cyanobacteria intact peptidoglycan is essential for cell envelope impermeability. Partial or total enzymic hydrolysis of peptidoglycan with lysozyme yields cells permeable to ions. 2-4 Although other means of permeabilization exist, 5,6 the enzymic method is superior when preservation of photosynthetic activity is desired. Different I H. Nikaido and M. Vaara, Microbiol. Rev. 49, 1 (1985). 2 j. Biggins, Plant Physiol. 42, 1442 (1967). 3 B. Ward and J. Myers, Plant Physiol. 50, 547 (1972). 4 G. C. Papageorgiou and T. Lagoyanni, Biochim. Biophys. Acta 807, 230 (1985). 5 B. Gerhardt and A. Trebst, Z. Naturforsch. B 20, 879 (1965). 6 S. J. Robinson, C. S. DeRoo, and C. F. Yocum, Plant Physiol. 70, 154 (1982).
METHODS IN ENZYMOLOGY. VOL. 167
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