A new high-performance liquid chromatographic method with evaporative light scattering detector for the analysis of phospholipids. Application to Iberian pig subcutaneous fat.

A new method for the analysis of phospholipids by normal-phase HPLC is described using a silica column. Addition of ammonia and triethylamine to a gradient based on chloroform/methanol/water promoted a good and rapid separation of phospholipid classes (20 min run). The use of an evaporative light scattering detector permitted an accurate analysis of a mixture of phospholipids. Calibration curves were linear within different range for each phospholipid class. The LOD and LOQ obtained were below 0.03 and 0.05 mg kg⁻¹ for all cases, respectively. Besides, a new method for the separation of phospholipids from total lipids before HPLC analysis by a solid-phase extraction (SPE) with Si cartridges has been developed. This methodology gave a good recovery ranging from 97 to 117%. The method was validated with a standard mixture of phospholipids. This method has been applied to characterize the phospholipid fraction of subcutaneous fat from Iberian pig. Cardiolipin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin have been described for first time in these samples. The fatty acid composition of the different phospholipid classes and their HPLC electrospray ionization mass spectrometry have been used for characterizing the molecular species present in each one.

[The structure of the glycerophosphate-containing O-specific polysaccharide from Escherichia coli 0130].

A phosphorylated O-specific polysaccharide was obtained by mild acidic degradation of the lipopolysaccharide from the intestinal bacterium Escherichia coli 0130 and characterized by the methods of chemical analysis, including dephosphorylation, and 1H and 13C NMR spectroscopy. The polysaccharide was shown to be composed of branched tetrasaccharide repeating units containing two N-acetyl-D-galactosamine residues, D-galactose, D-glucose, and glycerophosphate residues (one of each). The polysaccharide has the following structure, which is unique among the known bacterial polysaccharides.

Thermostabilization of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus.

Diglycerol phosphate accumulates under salt stress in the archaeon Archaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.

Altered composition of cerebral microvessel membrane phosphoglycerides from senescent mouse.

Phosphoglyceride and fatty acid composition was determined in the cellular membranes of isolated cerebral microvessels and brain parenchymal cells (neurons and glia) taken from 10-, 20-, and 27-30-month-old C57BL6/NNIA mice. Lipids were extracted from each fraction and the fatty acid profiles of ethanolamine, choline, serine, and inositol phosphoglycerides analyzed by gas chromatography. The results suggest that membrane phosphoglycerides from cerebral microvessels are significantly more affected by the aging process than are those of the brain parenchyma. Relative percentage for fatty acids in cerebral microvessels indicate an overall decline in membrane unsaturation with a concomitant elevation in the level of saturation. The decline in unsaturation is reflected primarily in the loss of precursor fatty acids for arachidonic (18:2n-6 and 20:3n-6) and docosahexaenoic (20:5n-3 and 22:5n-3) acids. Levels of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids in each phosphoglyceride remained unchanged with age; however, mol% for ethanolamine plasmalogen, a major source of these fatty acids, was significantly reduced in 27-30-month-old mice. Conversely, mol% for choline phosphoglyceride increased with age. The age-related changes in fatty acid profile for microvessel membrane phosphoglycerides are reflected by increased saturation/unsaturation ratios and decreased unsaturation indices. These parameters were not affected by aging in parenchymal membranes.

Identification of a putative tumor marker in breast and colon cancer.

Application of a recently developed phospholipid saponification procedure to malignant colon or breast tissue produces an unidentified phosphodiester resonance in the 31P nuclear magnetic resonance spectrum which appears to correlate with malignancy. Glycerol phosphodiesters were prepared from a malignant breast tumor by a Folch extraction of the tissue followed by saponification of the resulting phospholipids. These compounds were separated by ion exchange chromatography and the compound responsible for this new resonance was isolated and identified as glycerol 2-phosphoglycerol by high resolution nuclear magnetic resonance spectroscopy. Confirmation of this structure was achieved by chemical synthesis of glycerol 2-phosphoglycerol. Attempts to isolate the phospholipid responsible for this resonance showed that it is not derived from a new class of phospholipids but is most likely an artifact of the new saponification procedure used. Glycerol 2-phosphoglycerol is formed via glycerol 2,3-(cyclic) phosphate derived from phosphatidylcholine followed by reaction with glycerol. Malignant tissue is more prone to produce this compound than is nonmalignant tissue and the differences between these tissue types may be a form of phosphatidylcholine that is present in higher concentrations in malignant tumor membranes than in those of normal tissue.

Structure of the K24 antigen of E. coli O83:K24:H, a polymer that consists of alpha-Kdop and glycerol phosphate.

The structure of the K24 antigen of Escherichia coli O83:K24:H31 was elucidated by determination of composition and by 1H-, 13C-, and 31P-n.m.r. spectroscopy of the polymer and of a Kdo-glycerol (Gro) glycoside, obtained by mild alkaline hydrolysis and subsequent incubation with alkaline phosphatase. The K24 antigen has the repeating unit----7)-alpha-Kdop-(2----1)-Gro-(3-P. In the polymer, 56% of the repeating units are O-acetylated at C-4 of Kdo, approximately 28% at C-5 of Kdo, and approximately 16% are not acetylated.

Purification and characterization of two novel phosphoglycerides that modulate the glucocorticoid-receptor complex. Evidence for two modulator binding sites in the occupied/unactivated steroid hormone receptor.

Modulator is a novel ether aminophosphoglyceride that is commonly known as the low-molecular weight inhibitor of glucocorticoid-receptor complex activation. An ultra-large scale purification of modulator has been performed from 1000 rat livers. This purification was similar to our previous one (Bodine, P. V., and Litwack, G. (1988) J. Biol. Chem. 263, 3501-3512), but involved the chromatography of heated rat liver cytosol on a 7-liter bed volume Sephadex G-15 gel filtration column. Two peaks of modulator activity eluted from the giant gel-filtration column, and these two modulators (peak-1 and peak-2) were chromatographed separately on Dowex-1 anion-exchange columns. Both modulators were determined to be homogeneous after this step by analytical high-performance thin-layer chromatography, analytical high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy. Furthermore, although peak-1 and peak-2 differed in molecular weight, the two modulators co-chromatographed by anion-exchange, high-performance thin-layer, and high-performance liquid chromatography. These results suggest that the two modulators have similar structures and therefore appear to be isoforms of each other. In addition, both of the modulators are organic molecules that are devoid of molybdenum and 62 other metals. Activity assays indicated that the larger peak-1 modulator was five times more potent than the smaller peak-2 modulator at inhibiting receptor activation and at stabilizing the steroid-binding ability of the occupied and unoccupied receptors. Mixing experiments indicated that the activities of the two modulators were synergistic for both receptor activation inhibition and for occupied receptor steroid-binding stabilization. However, the effects of peak-1 and peak-2 modulator on unoccupied receptor steroid-binding stabilization were additive. Thus, although the two modulators have similar chemical structures, the biological potencies of the two compounds are different. Moreover, these results suggest that although the unoccupied/unactivated receptor has only one modulator binding site, the occupied/unactivated receptor has two modulator binding sites, one site for each of the isoforms.

'Lipoteichoic acid' of Bifidobacterium bifidum subspecies pennsylvanicum DSM 20239. A lipoglycan with monoglycerophosphate side chains.

The lipid macroamphiphile of Bifidobacterium bifidum subsp. pennsylvanicum DSM 20239 was extracted with phenol/water and purified by treatment with nucleases and hydrophobic interaction chromatography. From analytical data, the results of Smith degradation, hydrolysis with HF and methylation studies, the following structure is proposed: (formula; see text) where n and m are approximately 7-10 and 8-15, respectively. The monoglycerophosphate residues have the sn-glycero-1-phosphate configuration; 20-50% of them are substituted with L-alanine in ester linkage. The lipid anchor is most likely a galactosyldiacylglycerol, part of which carries a third fatty acid. This is the first example among gram-positive bacteria of a glycerophosphate-containing lipid macroamphiphile that carries the glycerophosphate residues as monomeric side chains on a lipoglycan. Further, it contains L-alanine in place of the D-alanine found in lipoteichoic acids.

Structural studies on the linkage unit between poly(N-acetylglucosamine 1-phosphate) and peptidoglycan in cell walls of Bacillus pumilus AHU 1650.

Structural studies were carried out on the polymer chains and their linkage regions in two kinds of teichoic acids, poly(N-acetylglucosamine 1-phosphate) [poly(GlcNAc-1-P)] and glycerol teichoic acid, bound to peptidoglycan in the cell walls of Bacillus pumilus AHU 1650. The poly(GlcNAc-1-P)-glycan complex isolated from lysozyme digests of the cell walls contained mannosamine and glycerol as minor components. On the basis of proton NMR spectroscopic data and isolation of N-acetylglucosamine 4-phosphate from acid hydrolysates, the poly(GlcNAc-1-P) was shown to be a polymer in which N-acetylglucosamine 1-phosphate units are joined at C-4 of the glucosamine residues. Mild alkaline hydrolysis of the poly(GlcNAc-1-P)-glycan complex gave a mannosamine-linked glycan fragment and the acidic polymer fraction that contained glycerol residues. Mild acid treatment of the mannosamine-linked glycan fragment gave the linkage disaccharide, ManNAc(beta 1----4)GlcNAc, whereas the acidic polymer fraction was degraded by this treatment into N-acetylglucosamine 4-phosphate and a glycerol-containing fragment characterized as P-(Gro-P)7 (Gro = glycerol). On the other hand, direct mild acid hydrolysis of the complex gave a fragment characterized as P-(Gro-P)7-ManNAc(beta 1----4)GlcNAc. These results lead to a conclusion that in the cell walls the poly(GlcNAc-1-P) chain is attached to peptidoglycan through a linkage unit, (Gro-P)7-ManNAc(beta 1----4)GlcNAc. By means of similar procedures, it was shown that the other cell wall polymer, glycerol teichoic acid, is also attached to peptidoglycan through the same disaccharide, ManNAc(beta 1----4)GlcNAc.

Structure of the Escherichia coli K2 capsular antigen. Stereochemical configuration of the glycerophosphate and distribution of galactopyranosyl and galactofuranosyl residues.

The Escherichia coli K2 capsular antigen is known to be composed of alpha-D-galactopyranosyl(1--2)glycerophosphate and alpha-D-galactofuranosyl(1--2)glycerophosphate units which are connected by phosphodiester bonds to C-4 of the galactopyranosyl and C-5 or C-6 of the galactofuranosyl moieties. In the present study the glycerophosphates were released by two different procedures and shown to have the sn-glycero-3-phosphate stereochemical configuration. In the first, the chain was fragmented by Smith degradation to glycerophosphothreitol from which the glycerophosphate was released by alkali hydrolysis. The structure-dependent low recovery of alpha-glycerophosphate (less than 10%) initiated the development of another degradative sequence which consisted of periodate oxidation, beta elimination, hydrazinolysis, and alkaline treatment. This way, approximately 90% of the glycerophosphate was released as sn-glycero-3-phosphate. beta elimination revealed in addition that most of the galactofuranosyl residues carry the phosphodiester bond at position 5. Separation by gel permeation chromatography and analysis of the fragments obtained by beta elimination showed that pyranosidic and furanosidic galactosyl residues alternate in the same chain and suggested the sequences Galf(p)GroP-(GalpGroP)n-Galf- and -GalfGroP-(GalpGroP)n-Galf-, where n is 6, 4, and 3, respectively.

Isocratic high-performance liquid chromatography separation of phosphoglycerides and lysophosphoglycerides.

An isocratic, rapid method for separation of phospho- and lysophospholipids by high-performance liquid chromatography employing acetonitrile, methanol and water as the mobile phase is described. Separation is achieved on a silica based cation-exchange column with individual components monitored directly by UV absorbance at 203 nm. In solutions of phospho- and lysophospholipid standards and in extracts of rabbit myocardium, recovery of individual components exceeds 95%. With the exception of phosphatidyl serine and phosphatidyl ethanolamine that could not be resolved other major phospho- and lysophospholipid constituents found in extracts of cell membranes are separated completely within 40 min. Retention time for selected moieties can be shortened even further with flow programming.

Studies on the relationship between glycerophosphoglycolipids and lipoteichoic acids. IV. Trigalactosylglycerophospho-acylkojibiosyldiacylglycerol and related compounds from Streptococcus lactis Kiel 42172.

Streptococcus lactis Kiel 42172 contains at least six unusually polar glycerophosphoglycolipids. The predominant one was composed of D-galactose, D-glucose, glycerol, acyl groups and phosphorus in a molar ratio of approx. 3 : 2 : 2 : 3 : 1. By analysis of the breakdown products of HF hydrolysis and Smith-degradation the structure was established to be [Galp (alpha 1 leads to 6)Galp(alpha 1 leads to 3)-sn-glycero(2 comes from 1 alpha Galp)-1-phospho] leads to 6Glcp(alpha 1 leads to 2), acyl leads to Glcp(alpha 1 leads to 3)-acyl2Gro. By HF hydrolysis the other compounds were shown to be in the main also derivatives of GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro but they released as water-soluble glycosides Gal(alpha 1 leads to 2)Gro, Gal(alpha 1 leads to 3)Gro, Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), Gal(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro and Gal(alpha 1 leads to 6)Gal-(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), respectively. In the lipid extract Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro and GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3) acyl2Gro were also observed. This set of compounds is proposed to constitute a biosynthetic series reflecting the individual steps in the synthesis of the lipoteichoic acid of Streptococcus lactis Kiel 42172 which is made up by the same lipid anchor and a non-classical poly(galabiosyl, galactosyl glycerophosphate)-chain (Koch, H.U. and Fischer, W. (1978) Biochemistry 17, 5275--5281).

Biosynthesis of glycosylated glycerolphosphate polymers in Streptococcus sanguis.

Two types of glycosylated glycerolphosphates were synthesized when a particulate enzyme prepared from Streptococcus sanguis was incubated with [3H]-phosphatidylglycerol and uridine diphosphate-[14C]glucose in the presence of MgCl2. The first type was extractable with saline and contained no fatty acid. The second type was pellet bound and could be extracted with 0.1% sodium dodecyl sulfate. Both types of polymers were purified and partially characterized. The first type of polymer was fractionated into five polymers, peaks 2a, 2b, 2c, 3a, and 3b. All except peak 2a, which contained only [3H]glycerol, contained both [3H]glycerol and [14C]glucose. [3H]NaBH4 reduction of acid hydrolysates of the polymers revealed that all of the polymers contained glucose as the major sugar componenta nd xylose as the minor sugar component. The second type of polymer was fractionated into three polymers, P-1, P-2, and P-3. All contained [3H]-glycerol, [14C]glucose, and fatty acids. P-1 appeared to be pure, whereas P-2 and P-3 contained two polymers each, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Characterization of an endogenous transcription inhibitor from Physarum polycephalum.

A substance has been purified from isolated nuclei of Physarum polycephalum by equilibrium and velocity gradient centrifugations, ion exchange chromatography and gel filtration which has a high molecular weight, can be labeled in vivo with 32P, is heat stable and resistant to amylases, proteases, nucleases and phosphodiesterase but is sensitive to phosphatases or hydrolysis. This material consists of phosphate and glycerol. It selectively inhibits in vitro transcription of RNA polymerases, predominantly the homologous enzyme A by binding to the enzyme. In the presence of this inhibitor of transcription a stable RNA polymerase-template complex cannot be formed. Binding to and inactivation of RNA polymerase is reversible at high ionic strength.

Complex phosphoglycerides from rat liver outer mitochondrial membranes.

The ethanolic extract from rat liver mitochondrial membranes contains a number of highly polar complex lipids, which are found in the aqueous layer when subjected to the usual chloroform-water partition procedures. Two of these lipids have been purified by thin-layer chromatography, and their structures partially elucidated. Apparently, both are derivatives of phosphatidylglycerol, with a bulky polar head containing sugars and aminoacids. They are specifically located in the outer membrane. Several structures are suggested, as well as a possible functional rôle for these compounds.

[Hemoglobins, XXI: sequence analysis of porcine hemoglobin (author's transl)]. / Hämoglobine, XXI. Die Sequenzanalyse des Hämoglobins des Schweines.

The hemoglobin of a bavarian domestic pig (Suidae) was isolated. The chains were separated by countercurrent distribution, then cleaved with trypsin. The isolated peptides were sequenced with hydrophilic phenylisothiocyanate I and IV, or with a dimethylaminopropyne program in the sequenator. The sequences of the chains are given. Some methodical aspects of automatic sequencing are discussed and the sequences of human porcine hemoglobin are compared. The role of adult pig haemoglobin as foetal hemoglobin is discussed.

The linkage of sugar phosphate polymer to peptidoglycan in walls of Micrococcus sp. 2102.

1. Protein-free walls of Micrococcus sp. 2102 contain peptidoglycan, poly-(N-acetylglucosamine 1-phosphate) and small amounts of glycerol phosphate. 2. After destruction of the poly-(N-acetylglucosamine 1-phosphate) with periodate, the glycerol phosphate remains attached to the wall, but can be removed by controlled alkaline hydrolysis. The homogeneous product comprises a chain of three glycerol phosphates and an additional phosphate residue. 3. The poly-(N-acetylglucosamine 1-phosphate) is attached through its terminal phosphate to one end of the tri(glycerol phosphate). 4. The other end of the glycerol phosphate trimer is attached through its terminal phosphate to the 3-or 4-position of an N-acetylglucosamine. It is concluded that the sequence of residues in the sugar 1-phosphate polymer-peptidoglycan complex is: (N-acetylglucosamine 1-phosphate)24-(glycerol phosphate)3-N-acetylglucosamine 1-phosphate-muramic acid (in peptidoglycan). Thus in this organism the phosphorylated wall polymer is attached to the peptidoglycan of the wall through a linkage unit comprising a chain of three glycerol phosphate residues and an N-acetylglucosamine 1-phosphate, similar to or identical with the linkage unit in Staphylococcus aureus H.