Masateru Nishihara

Lipid molecular species composition of thylakoid membranes

Lipid molecular species compositions of chloroplast thylakoid membranes of mesophyll cells from Spinacia oleracea, Glycine max, Oryza sativa and Zea mays and of bundle sheath cells from Zea mays have been quantitatively determined. No significant difference in the lipid molecular species composition was found among the five membrane sources. The predominant molecular species of monogalactosyldiacylglycerol was the 1-linolenoyl parallel to 2-linolenoyl species. The 1-linolenoyl parallel to 2-linoenoyl and 1-palmitoyl parallel to 2-linolenoyl species were the major molecular species of digalactosyldiacylglycerol. 6-Sulfoquinovosyldiacylglycerol was mainly composed of the palmitoyl parallel to linolenoyl and palmitoyl parallel to lineolyl species. Almost all of the C-2 position of phosphatidylglycerol were esterified with the palmitoyl or delta 3-trans-hexadecenoyl residue. The molecular species compositions of phosphatidylcholine and phosphatidylinositol were basically similar to those of membranes in non-photosynthetic tissues.

Relation between growth temperature of E. coli and phase transition temperatures of its cytoplasmic and outer membranes.

Cells of wild-type E. coli B were grown at 17, 27 and 38 degrees C, and their cell membranes were fractionated into the cytoplasmic and the outer membranes. Chemical assay proved that the molar ratio of saturated to unsaturated fatty acids increases in phospholipids extracted from each membrane as the growth temperature increases. The transition temperature at which the solid phase disappears was determined by X-ray diffraction in these biomembranes and also membranes of extracted phospholipids and of extracted lipopolysaccharide. The transition temperatures of the cytoplasmic membrane and of the membranes of phospholipids extracted from the cytoplasmic and the outer membranes increased with the growth temperature in good parallelism to the molar ratio of saturated to unsaturated fatty acids. The transition temperature of the outer membrane was less sensitive to the growth temperature, presumably due to the presence of lipopolysaccharide. The transition temperature of the membranes of lipopolysaccharide extracted from the outer membrane was 25 degrees C, for the cells grown at 27 and 37 degrees C. For the cells grown at 17 degrees C, the extracted lipopolysaccharide gave a broad diffraction peak and did not exhibit a solid-fluid phase transition between --5 and 40 degrees C.

Structural analysis by reductive cleavage with LiAlH4 of an allyl ether choline-phospholipid, archaetidylcholine, from the hyperthermophilic methanoarchaeon Methanopyrus kandleri

A choline-containing phospholipid (PL-4) in Methanopyrus kandleri cells was identified as archaetidylcholine, which has been described by Sprott et al. (1997). The PL-4 consisted of a variety of molecular species differing in hydrocarbon composition. Most of the PL-4 was acid-labile because of its allyl ether bond. The identity of PL-4 was confirmed by thin-layer chromatography followed by positive staining with Dragendorff reagent and fast-atom bombardment-mass spectrometry. A new method of LiAlH4 hydrogenolysis was developed to cleave allyl ether bonds and recover the corresponding hydrocarbons. We confirmed the validity of the LiAlH4 method in a study of the model compound synthetic unsaturated archaetidic acid (2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate). Saturated ether bonds were not cleaved by the LiAlH4 method. The hydrocarbons formed following LiAlH4 hydrogenolysis of PL-4 were identified by gas-liquid chromatography and mass spectrometry. Four kinds of hydrocarbons with one to four double bonds were detected: 47% of the hydrocarbons had four double bonds; 11% had three double bonds; 14% had two double bonds; 7% had one double bond; and 6% were saturated species. The molecular species composition of PL-4 was also estimated based on acid lability: 77% of the molecular species had two acid-labile hydrocarbons; 11% had one acid-labile and one acid-stable hydrocarbon; and 11% had two acid-stable hydrocarbons. To our knowledge, this is the first report of a specific chemical degradation method for the structural analysis of allyl ether phospholipid in archaea.

Quantitative determination of methanogenic cells based on analysis of ether-linked glycerolipids by high-performance liquid chromatography

A method for the quantitative determination of methanogenic cells in natural environments is described. The method is based on the determination of ether-linked glycerolipids that is specific to methanogens and other archaebacteria. Core lipids (archaeol and caldarchaeol), prepared from total lipids by a combination of acetolysis and acid methanolysis, were quantitated as the corresponding dinitrobenzoyl derivatives by high-performance liquid chromatography. The total amount of core lipids was proportional to the amount of methanogenic cells. This method has high sensitivity, and the lowest limit of detection of methanogenic cells was 2.5 μ g (dry weight) in a 500- μ l sample of culture or sludge. An identical standard curve was obtained for Methanobacterium thermoautotrophicum and Methanospirillum hungatei . With this method, it was possible to estimate methanogenic cells in a sample of sludge from an anaerobic digestor. In spite of contamination by large amounts of multifarious adulterants in the sludge, 83% of M. thermoautotrophicum cells added to the sludge was recovered and quantitated by this method.

Inference of methanogenic bacteria in wastewater digestor sludges by lipid component analysis

The presence of methanogens of the genera Methanosarcina and Methanosaeta, and the family Methanomicrobiaceae, and the absence of the family Methanobacteriaceae were inferred from the analysis of lipid component parts in three kinds of sludge from two anaerobic digestors by comparison with the distribution of the lipid component parts among 31 species of methanogens previously reported.

Expression and use of Methanobacterium thermoautotrophicum sn-glycerol 1-phosphate dehydrogenase for the assay of sn-glycerol 1-phosphate in Archaea

Abstract sn -glycerol-1-phosphate (G-1-P) dehydrogenase is the key enzyme for biosynthesis of the enantiomeric glycerophosphate backbone of ether phospholipids of Archaea. The gene encoding this enzyme in Methanobacterium thermoautotrophicum ( egsA ) was used to construct an expression plasmid pTrcG1Pdh for Escherichia coli . The G-1-P dehydrogenase activity of E. coli XL1-blue/pTrcG1Pdh was maximal 8–10 h after induction. The expressed G-1-P dehydrogenase was purified 4300-fold from the soluble fraction to homogeniety after 4300 times purification by a procedure consisting of fractionation with ammonium sulfate precipitation, and hydrophobic and ion-exchange chromatographies. The yield was about 70%. The V max value for the forward reaction to produce G-1-P was 740 units (μmol/min)/mg, with a K m of 0.21 mM for NADH and 0.39 mM for dihydroxyacetone phosphate. The K m s for G-1-P and NAD + in the backward reaction were 10.5 and 0.46 mM, respectively. These kinetic constants are similar to those for the enzyme from M. thermoautotrophicum . G-1-P dehydrogenase was successfully used to analyze the stereospecificity of glycerophosphate, which is an intermediate of phospholipid biosynthesis and glycerol metabolism; the rate of NADH formation was proportional to the G-1-P concentration up to 3 mM in the presence of 0.02 unit of the purified enzyme.

Phospholipids of membranes of cultured cells and the products of protoplast fusion

Abstract The variety and distribution of phospholipids in the cell membranes of cultured cells and their fatty acid composition were analysed. Membranes of suspension cultured cells of Rauwolfia serpentina var. Bentham, Nicotiana tabacum var. Samsun, Atropa belladonna and Bouvardia ternifolia had almost the same composition of phosphatidylcholine, PC ( ca 50%); phosphatidylethanolamine, PE ( ca 25%); phosphatidylinositol, PI ( ca 10%); phosphatidylglycerol, PG (several %); and phosphatidic acid, PA (several %). We determined the distribution of the molecular species of the three major phospholipid fractions (PC, PE and PI) in R. serpentina and N. tabacum . Membranes of both cell-types contained basically similar molecular species, 1–16:0/2–18:2 the main type, particularly in the PC- and PE- fractions; 1–18:2/2–18:2 and 1–18:0/2–18:2 for R. serpentina ; and 1–16:0/2–18:3, 1-18:0/2-18:3 and 1-18:2/2-18:2 for N. tabacum . The influence of membrane fluidity on protoplast fusion as effected by the phase transition of the phospholipids, is discussed.