Lipid fingerprints of intact viruses by MALDI-TOF/mass spectrometry.

A number of viruses contain lipid membranes, which are in close contact with capsid proteins and/or nucleic acids and have an important role in the viral infection process. In this study membrane lipids of intact viruses have been analysed by MALDI-TOF/MS with a novel methodology avoiding lipid extraction and separation steps. To validate the novel method, a wide screening of viral lipids has been performed analysing highly purified intact bacterial and archaeal viruses displaying different virion architectures. Lipid profiles reported here contain all lipids previously detected by mass spectrometry analyses of virus lipid extracts. Novel details on the membrane lipid composition of selected viruses have also been obtained. In addition we show that this technique allows the study of lipid distribution easily in subviral particles during virus fractionation. The possibility to reliably analyse minute amounts of intact viruses by mass spectrometry opens new perspectives in analytical and functional lipid studies on a wider range of viruses including pathogenic human ones, which are difficult to purify in large amounts.

Natronococcus roseus sp. nov., a haloalkaliphilic archaeon from a hypersaline lake.

A novel halophilic archaeon, strain CG-1(T), belonging to the genus Natronococcus was isolated from sediment of the soda lake Chagannor in Inner Mongolia, China. The colonies of this strain were pink pigmented, the intensity of the colour decreased when the cells grew at salt saturation levels. The cells were non-motile cocci and strictly aerobic. Hypotonic treatment did not cause cell lysis, even in distilled water. Strain CG-1(T) grew at 15-30.0 % (w/v) NaCl and at 30-50 °C and pH 8.0-11.0, with optimal growth occurring at 25-30 % (w/v) NaCl, 37-45 °C and pH 9-9.5. MgCl(2) was not required for growth. Strain CG-1(T) was most closely related to the type strains of Natronococcus amylolyticus Ah-36(T), Natronococcus jeotgali B1(T) and Natronococcus occultus SP4(T), with which it shared 98.4 %, 96.2 and 95.7 % 16S rRNA gene sequence similarity, respectively. The polar lipids consisted of C(20)C(20) and C(20)C(25) derivatives of phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and minor phospholipid components. No glycolipids were detected. The DNA G+C content of strain CG-1(T) was 62.1 mol%. DNA-DNA hybridization with N. amylolyticus DSM 10524(T), phylogenetically the most closely related species, was 39 %; this value showed that strain CG-1(T) constituted a different genospecies. The comparison of 16S rRNA gene sequences, detailed phenotypic characterization, polar lipid profile and DNA-DNA hybridization studies revealed that strain CG-1(T) belongs to the genus Natronococcus and constitutes a novel species for which the name Natronococcus roseus sp. nov. is proposed. The type strain is CG-1(T) (=CECT 7984(T)=IBRC-M 10656(T)=JCM 17958(T)).

Isolation and characterization of lipids strictly associated to PSII complexes: focus on cardiolipin structural and functional role.

In this work, lipid extracts from spinach membrane fragments enriched in Photosystem II (PSII) and from spinach PSII dimers were analyzed, by means of Thin Layer Chromatography (TLC) and Electro-Spray Ionization Mass Spectrometry. Cardiolipin found in association with PSII was isolated and purified by preparative TLC, then characterized by mass and mass-mass analyses. Cardiolipin structures with four unsaturated C18 acyl chains and variable saturation degrees were evidenced. Structural and functional effects of different phospholipids on PSII complexes were investigated by Fluorescence, Resonance Light Scattering and Oxygen Evolution Rate measurements. An increment of PSII thermal stability was observed in the presence of cardiolipin and phosphatidylglycerol.

Lipid content in higher plants under osmotic stress.

In this work, we performed investigations on the lipid content of higher plants (spinach) under hyperosmotic stress, by means of thin layer chromatography (TLC) and mass spectrometry. In particular, the experiments have been performed at different plant organization levels: whole leaves, freshly prepared protoplast suspension and mesophyll cells obtained by reformation of the cell wall from protoplast suspension. The results obtained showed that hyperosmotic stress induces changes in the phospholipid content depending on the different plant organization levels studied. All phospholipids showed an increment of their content in stressed whole leaves. In particular, phosphatidylglycerol (PG) redoubles its content by 1 h of osmotic shock. Different responses to hyperosmotic stress were reported for the other systems. In the case of protoplasts, an increment of PG, phosphatidylcholine (PC) and phosphatidylinositol (PI) together with biphosphatidylglycerol (BPG) and phosphatidylethanolamine (PE) content decreasing were observed in stressed sample. For PG, identified as PG (34:4) by elecrospray ionization mass spectrometry, the increment was of about 30%. In the case of cells, conversely, a decrease of PG content under osmotic stress was recorded. The results suggest an important role of phospholipids, in particular of PG, in the osmotic stress response.

The role of potassium and chloride ions on the Na+/acidic amino acid cotransport system in rat intestinal brush-border membrane vesicles.

The Na+/L-glutamate (L-aspartate) cotransport system present at the level of rat intestinal brush-border membrane vesicles is specifically activated by the ions K+ and Cl-. The presence of 100 mM K+ inside the vesicles drastically enhances the uptake rate and the transient intravesicular accumulation (overshoot) of the two acidic amino acids. It has been demonstrated that the activation of the transport system depended only in the intravesicular K+ concentration and that in the absence of any sodium gradient, an outward K+ gradient was unable to influence the Na+/acidic amino acid transport system. It was also found that Cl- could specifically activate the Na+-dependent L-glutamate (L-aspartate) uptake either in the presence or in the absence of K+. Also the effect of Cl- was observed only in the presence of an inward Na+ gradient and it was noted to be higher when chloride ion was present on both sides of the membrane vesicles. No influence (activation or accumulation) was observed in the absence of the Na+ gradient and in the presence of chloride gradient. L-Glutamate uptake measured in the presence of an imposed diffusion potential and in the presence of K+ or Cl- did not show any translocation of net charge.

Chloride dependence of the sodium-dependent glycine transport in pig kidney cortex brush-border membrane vesicles.

The Na+-dependent glycine uptake in pig kidney cortex brush-border membrane vesicles is specifically enhanced by the presence of Cl-. The Na+-independent glycine uptake is not affected by Cl-. Various anions tested could not substitute Cl- in the activation of the Na+-dependent glycine transport. Cl- is specifically required on the outer membrane side. The Na+-dependent glycine uptake is higher in the presence of an inwardly directed Cl- gradient than the one measured in the presence of equilibrated Cl-. The Na+-dependent glycine uptake depends on, and is saturable at increasing Cl- concentrations. By studying the activation of glycine uptake by Na+ in the presence and in the absence of Cl-, evidence was found that two different Na+-dependent glycine transport pathways are present in pig kidney cortex brush-border membrane vesicles. The kinetics of the glycine uptake measured in the presence of an inwardly directed NaCl gradient show the presence of two glycine transport systems, a low-affinity, high-capacity one and a high-affinity, low capacity one. In the absence of Cl- the high-affinity, low-capacity transport is almost suppressed, thus indicating the presence of a high-affinity glycine transport system simultaneously dependent on both Na+ and Cl- ions.

Palmitic acid is associated with halorhodopsin as a free fatty acid. Radiolabeling of halorhodopsin with 3H-palmitic acid and chemical analysis of the reaction products of purified halorhodopsin with thiols and NaBH4.

Halorhodopsin, isolated from Halobacterium salinarium cells incubated with tritiated palmitic acid, co-elutes with labeled palmitate in phenylsepharose CL-4B chromatography. Halorhodopsin-bound 3H-palmitate is not readily displaced by prolonged exposure to a large excess of detergents and by re-chromatography of radiolabeled halorhodopsin on phenylsepharose. On other hand, the association of labeled palmitate with purified halorhodopsin is not resistant to denaturation induced either by isopropanol/hexane or by SDS gel electrophoresis. We have tested the hypothesis that tightly associated palmitate is bound to halorhodopsin through a thioester bond, which is unstable in denaturing conditions. Using GC/MS, we have analysed the reaction products of native halorhodopsin with specific thioester reagents, thiols and NaBH4, which are inactive on free fatty acids. The results of this analytical approach indicate that there is no thioester bond between halorhodopsin and palmitic acid and that palmitic acid is associated with halorhodopsin as a free fatty acid.

Electroneutral Na+/dicarboxylic amino acid cotransport in rat intestinal brush border membrane vesicles.

L-Glutamate and L-aspartate transport into osmotically active intestinal brush border membrane vesicles is specifically increased by Na+ gradient (extravesicular greater than intravesicular) which in addition energizes the transient accumulation (overshoot) of the two amino acids against their concentration gradients. The "overshoot" is observed at minimal external Na+ concentration of 100 mM for L-glutamate and 60 mM for L-aspartate; saturation with respect to [Na+] was observed at a concentration near 100 mM for both amino acids. Increasing amino acid concentration, saturation of the uptake rate was observed for L-glutamate and L-aspartate in the concentration range between 1 and 2 mM. Experiments showing mutual inhibition and transtimulation of the two amino acids indicate that the same Na+ -dependent transport system is shared by the two acidic amino acids. The imposition of diffusion potentials across the membrane vesicles artificially induced by addition of valinomycin in the presence of a K+ gradient supports the conclusion that the cotransport Na+/dicarboxylic amino acid in rat brush border membrane vesicles is electroneutral.

Role of palmitic acid on the isolation and properties of halorhodopsin.

Purified halorhodopsin was isolated from Halobacterium halobium as previously described (Duschl, A. et al. (1988) J. Biol. Chem. 263, 17016-17022). Two purple bands were eluted from phenyl-Sepharose column, indicating the presence of differently retained halorhodopsin forms; the absorption spectra of the two halorhodopsin bands in the dark were not different. By gas chromatography/mass spectrometry we could identify palmitate (which is only a minor lipid component of archaeal cells) among lipids associated with purple fractions. Typically the palmitate content of the first eluted band was higher than that of the second, indicating a correlation between the palmitate content and the retention time; from one to two fatty acid molecules per halorhodopsin molecule were present depending on the fraction analysed. Very little or no palmitate was released from denatured halorhodopsin. By adding palmitate to buffers used in the phenyl-Sepharose chromatography, only one sharp purple band was collected, corresponding to the less retained halorhodopsin fraction. Pentadecanoic fatty acid could also affect the halorhodopsin chromatography. Chromatography of halorhodopsin in the presence of beta-mercaptoethanol showed only one band, corresponding to the more retrained halorhodopsin form. The two halorhodopsin fractions had different photoreactivity; the less retained halorhodopsin fraction (at higher palmitate content) showed an higher rate of decay of the absorbance at 570 nm upon illumination. By following the decay of the absorbance at 570 nm upon addition of alkali in the dark, we found that the two halorhodopsin fractions had different pKa values of deprotonation.

Enrichment of cardiolipin content throughout the purification procedure of photosystem II.

Photosystem II is a multisubunit membrane complex which performs the water oxidation process in the higher plants. Core dimers and monomers of photosystem II have been isolated from thylakoid membranes by sucrose density gradient centrifugation. Lipids extracted from different photosystem II-enriched fractions obtained from spinach thylakoids have been analysed by thin layer chromatography. Cardiolipin is enriched throughout the purification of photosystem II complexes; in particular dimers contained two times more cardiolipin than their monomeric counterparts.

Role of endogenous lipids in the chromophore regeneration of bacteriorhodopsin.

The regeneration method of Khorana [J. Biol. Chem. 262 (1987) 9271] has been modified in order to study the effect of endogenous archaeabacterial lipids and, in particular, of glycocardiolipin (GlyC) in the refolding and chromophore regeneration of bacteriorhodopsin (BR). BR refolding and chromophore regeneration could be obtained in the presence of endogenous lipid mixtures containing or not containing glycocardiolipin; however, the kinetics of bacteriorhodopsin regeneration in the presence of glycocardiolipin was faster than in its absence. These results show for the first time that the interaction of glycocardiolipin with bacteriorhodopsin favours its refolding from the denaturated state and the chromophore regeneration.

Hybrid charge transfer complexes based on archaeal glycolipids wrapping single walled carbon nanotubes.

SWNTs have been functionalized with an archaeal glycolipid which wraps around the nano-objects in a single layer or bilayer, as a function of the nanotube diameter. Hydrogen bonds between the lipid glucose rings and the aromatic SWNT walls are involved in the formation of hybrid complexes resulting in electron transfer from the glycolipid to the nanotubes.

Role of phospholipids in the binding of bumetanide to the rabbit parotid Na/K/Cl cotransporter.

It was recently reported (Turner, R.J., George, J.N., 1990, J. Membrane Biol. 113:203-210) that the high affinity bumetanide binding site of the rabbit parotid Na/K/Cl cotransporter could be extracted from a basolateral membrane preparation from this gland using relatively low concentrations of the non-ionic detergent Triton X-100. At the detergent:protein ratios required for complete membrane solubilization bumetanide binding activity in this extract was lost but could be recovered by the addition of crude soybean lipids. In the present paper the ability of various purified lipids to restore high affinity bumetanide binding activity in detergent solubilized rabbit parotid basolateral membranes is studied. We show that the effect of exogenous lipid on the detergent-inactivated bumetanide binding site is to increase the affinity of binding without affecting the number of binding sites. Of the 11 lipid species tested, several relatively minor, negatively charged membrane phospholipids are the most effective in restoring binding activity (phosphatidylserine approximately phosphatidylglycerol greater than phosphatidylinositol greater than cardiolipin), while the major mammalian plasma membrane lipid components phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol are without effect. In addition, we show that in the presence of these minor lipids the affinity of bumetanide binding is considerably increased over that observed in the native membrane (e.g., Kd approximately 0.06 microM in membranes extracted with 0.3% Triton and treated with 0.15% wt/vol phosphatidylserine, vs. Kd approximately 3 microM in native basolateral membranes). This dramatic dependence of bumetanide binding affinity on the presence of certain lipid species suggests that the properties of the bumetanide binding protein in situ may be quite dependent on the minor lipid content of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

[Uptake of L-(+)lactate by cell membrane (luminal and contraluminal) isolated from rat small intestine microvilli]. / Uptake di L-(+)lattato in membrane plasmatiche (luminali e contraluminali) isolate de enterociti di intestino tenue di ratto.

L-lactate uptake was measured in vesicles formed by intestinal brush border and baso-lateral membranes, using a rapid filtration technique. In the presence of a Na+ gradient directed into the vesicle, L-lactate can be transiently accumulated in brush border vesicles, but not in baso-lateral ones. The transient L-lactate accumulation does not occur in the presence of a KCl gradient. alpha-cyanocinammic acid strongly inhibits L-lactate uptake in brush border vesicles, but not in baso-lateral ones. These results support the existence of a carrier mediated, Na+ dependent, transport of L-lactate across the brush border membrane.

Mechanism of Cl- transport in eel intestinal brush-border membrane vesicles.

Cl- transport was studied in a preparation of brush-border membrane vesicles (BBMV) from seawater eel intestine. 36Cl- uptake appeared to be stimulated by a positive inside membrane diffusion potential generated (a) by a concentration gradient of salts, the cations of which are more permeable than the anions, (b) by a K+ diffusion potential obtained by imposing a K+ concentration gradient (Cout greater than Cin) in the presence of valinomycin, (c) an inwardly directed H+ ion concentration gradient. The membrane-potential-driven Cl- transport was inhibited by 1 mM 5-nitro-2-(4-phenylpropyl-amino)-benzoic acid. Arachidonic acid also inhibited Cl- uptake in eel intestinal BBMV, but the effect appeared to be unspecific, as the unsaturated fatty acid also affected the Na+ dependent D-glucose uptake. The effect of arachidonic acid was reversed in the presence of bovine serum albumin. Cl- influx was the same in the presence of inwardly directed gradients of Li+, Na+ or K+, arguing against the presence of Na(+)-Cl-, as well as K(+)-Cl- cotransport. The absence of a significant contribution of the Na(+)-K(+)-2Cl- cotransport mechanism to the Cl- uptake in seawater eel intestinal BBMV was indicated from the observations that Cl- uptake was not stimulated by the simultaneous presence of inwardly directed Na+ and K+ gradients, and that it was nearly insensitive to 1 mM bumetanide in the presence of extravesicular Na+ and K+. Furthermore, no evidence for the dependence of Cl- uptake on the Na+ gradient was obtained under a short-circuited membrane diffusion potential, i.e. in the presence of equilibrated K+ and valinomycin.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of 3-deacetyl cephalosporins by Aspergillus niger lipase.

Lipase from Aspergillus niger was used for the selective hydrolysis of the 3-O-acetate of cephalosporin C to give an intermediate useful for further chemical elaborations. This lipase was purified to homogeneity and its properties compared with previously published data that present some discrepancies. The lipase proved to be very effective in catalyzing 3-O-acetate hydrolysis and versatile toward substitution on the beta-lactamic ring. In fact, as an example, two other cephalosporinic derivatives, cephalotin and cefotaxime, were efficiently deacetylated. The lipase was immobilized on Eupergit C and employed continuously in either a column or a batch reactor for 2 months without appreciable loss of activity. (c) 1996 John Wiley & Sons, Inc.

[Glycine uptake in brush border vesicles isolated from the rat intestinal cells]. / Uptake di glicina in vescicolf di orletto a spazzola isolate da enterociti di ratto.

The uptake of glycine in osmotically active brush border membrane vesicles (obtained by the Mg++ precipitation method) has been studied and a partial characterization of its transport system has been established. The glycine uptake in these vesicles was stimulated by the presence of sodium and in the presence of an inwardly directed Na+ -gradient glycine was accumulated inside the vesicles. The effect of Na+ was specific; other monovalent cation as Li+, K+, Rb+ and choline were uneffective in the stimulation of glycine uptake, under the same experimental conditions. Preliminary experiments show an important role of some anions on the glycine uptake. A strong inhibition in the uptake rate was found when the measurements were carried out in the presence of sodium cyclamate, while in the presence of NaSCN the measured uptake values were similar to those observed in the presence of NaCl.