Sequence specific stabilization of a linear analog of the antifungal lipopeptide iturin A2 by sodium during low energy electrospray ionization mass spectrometry conditions.

The structures and stability of sodiated species of 8-Beta, a linear lipopeptide analog (beta-aminotetradecanoyl-NYNQPNS) of the antifungal peptide iturin A2, were evaluated by electrospray ionization mass spectrometry (ESI-MS). Association of the lipopeptide, 8-Beta, with sodium afforded protection from fragmentation at high cone voltages and increasing collision energy conditions in the ESI-MS. The order of decreasing stability was found as 8-Beta 1Na > 8-Beta 2Na > 8-Beta 3Na > 8-Beta. Substantial differences were found between fragmentation patterns of the free and sodiated molecular species. Breakage of the N-terminal peptide bond of L-Pro generated the major product ions of the free 8-Beta parent ion. Impaired fragmentation of the sodium adducts of 8-Beta, indicated that this bond is protected by sodium complexation. Fragmentation patterns of the sodiated lipopeptide further revealed two specific binding sites for a nonsolvated sodium ion within the two type II beta-turn sequences (beta-aminotetradecanoyl-NYN and QPNS) of the natural iturin A2. It is proposed that specific interaction with sodium takes place with most of the peptide bond oxygens in these turns, and with the Gln sidechain. This interaction leads to stabilized structures in which the peptide backbone, specifically the peptide bonds in which L-Pro participates, is protected against low-energy fragmentation during ESI-MS.

Quantification of plasma membrane ergosterol of Saccharomyces cerevisiae by direct-injection atmospheric pressure chemical ionization/tandem mass spectrometry.

A method for the quantification of ergosterol by atmospheric pressure chemical ionization (APcI) mass spectrometry with direct injection is described. Ergosterol and squalene were ionizable with methanol as the carrier solvent. Using positive-mode tandem mass spectrometry (MS/MS), ergosterol could be identified unambiguously without interference from structurally related compounds such as lanosterol, cholesterol, and squalene. Molecular ions of ergosterol, lanosterol, and cholesterol were detected as the [M + H - H(2)O](+) ion species, while squalene appeared as the [M + H](+) ion species. Upon fragmentation of the three sterols and squalene, the product ion at m/z 69 was present as one of the major fragments in all four compounds. This product ion was used for the quantification of ergosterol in multiple-reaction-monitoring acquisition mode. The relationship between signal intensity and ergosterol concentration was linear over the concentration range of 0.15 to 5 microg/ml, or 7. 56-252 pmol ergosterol per 20 microl injection. The plasma membrane ergosterol of the yeast Saccharomyces cerevisiae could be quantified reproducibly without the need for prior separation from other lipids or derivatization. Six repeated injections of ergosterol standards at concentrations of 0.95 and 4.25 microg/ml gave standard deviations of 0.031 and 0.084, respectively, and coefficients of variation of 3.33 and 1.98%, respectively. The coefficient of variation for the four independently extracted membrane ergosterol samples was 11.18%. The presence of other lipids in a crude lipid extract did not interfere with the ergosterol determination. Direct injection APcI with multiple reaction monitoring is aconvenient and sensitive method for ergosterol quantification requiring no prior fractionation.

Implications of FPS1 deletion and membrane ergosterol content for glycerol efflux from Saccharomyces cerevisiae.

The deletion of the gene encoding the glycerol facilitator Fps1p was associated with an altered plasma membrane lipid composition in Saccharomyces cerevisiae. The S. cerevisiae fps1delta strain respectively contained 18 and 26% less ergosterol than the wild-type strain, at the whole-cell level and at the plasma membrane level. Other mutants with deficiencies in glycerol metabolism were studied to investigate any possible link between membrane ergosterol content and intracellular glycerol accumulation. In these mutants a modification in intracellular glycerol concentration, or in intra- to extracellular glycerol ratio was accompanied by a reduction in plasma membrane ergosterol content. However, there was no direct correlation between ergosterol content and intracellular glycerol concentration. Lipid composition influences the membrane permeability for solutes during adaptation of yeast cells to osmotic stress. In this study, ergosterol supplementation was shown to partially suppress the hypo-osmotic sensitivity phenotype of the fps1delta strain, leading to more efficient glycerol efflux, and improved survival. The erg-1 disruption mutant, which is unable to synthesise ergosterol, survived and recovered from the hypo-osmotic shock more successfully when the concentration of exogenously supplied ergosterol was increased. The results obtained suggest that a higher ergosterol content facilitates the flux of glycerol across the plasma membrane of S. cerevisiae cells.

The interaction of analogues of the antimicrobial lipopeptide, iturin A2, with alkali metal ions.

Electrospray mass spectrometry was employed as a tool in this first study on the molecular interaction between the alkali metal ions and antifungal lipopeptide iturin A, and some analogues. Cationisation by sodium and signal intensity of lipopeptide species depended on sodium concentration, but was independent of sample solvent, carrier solvent polarity and sample pH between 4 and 11. 8-Beta, a linear analogue of iturin A2 (8-Beta; beta-aminotetradecanoyl-NYNQPNS), and its shorter linear lipopeptide analogues, associated either one or two alkali metal cations, while the N-->C cyclic peptides associated with only one cation. The chirality of the beta-NC14 residue had a limited influence on the cationisation. It was observed that 8-Beta contained at least four interaction sites for a cation of which two, the C-terminal carboxylate and the side-chain of tyrosine, can take part in ionic interaction with a cation. It is proposed that the remaining two interaction centres of alkali metal ions are within the two type II beta-turns found in conformation of natural iturin A. This was corroborated by the diminished capacity of the shorter peptides, in which one of the beta-turns was eliminated to bind a second larger cation. All the lipopeptides showed the same order of alkali metal ion selectivity: Na+ > K+ > Rb+. These results indicated a size limitation in the interaction cavity or cavities. The absence of, or observation of only low abundance, di-cationised complexes of cyclic peptides the indicated association of the cation in the interior of the peptide ring. It is thus hypothesised that alkali metal ions can bind in one of the two beta-turns in the natural iturin A molecule.

Branched-chain alpha-keto acid catabolism via the gene products of the bkd operon in Enterococcus faecalis: a new, secreted metabolite serving as a temporary redox sink.

Recently the bkd gene cluster from Enterococcus faecalis was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain alpha-keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified the presence of a single promoter upstream of the bkd gene cluster. Furthermore, a putative catabolite-responsive element was identified in the promoter region, indicative of catabolite repression. Consistent with this was the observation that expression of the bkd gene cluster is repressed in the presence of glucose, fructose, and lactose. It is proposed that the conversion of the branched-chain alpha-keto acids to the corresponding free acids results in the formation of ATP via substrate level phosphorylation. The utilization of the alpha-keto acids resulted in a marked increase of biomass, equivalent to a net production of 0.5 mol of ATP per mol of alpha-keto acid metabolized. The pathway was active under aerobic as well as anaerobic conditions. However, under anaerobic conditions the presence of a suitable electron acceptor to regenerate NAD(+) from the NADH produced by the branched-chain alpha-keto acid dehydrogenase complex was required for complete conversion of alpha-ketoisocaproate. Interestingly, during the conversion of the branched-chain alpha-keto acids an intermediate was always detected extracellularly. With alpha-ketoisocaproic acid as the substrate this intermediate was tentatively identified as 1, 1-dihydroxy-4-methyl-2-pentanone. This reduced form of alpha-ketoisocaproic acid was found to serve as a temporary redox sink.

Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071.

The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100 degrees C, 60 min; 50% of activity remained after 15 min at 121 degrees C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the alpha and beta peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.

Positive and negative modulation of H-ras transforming potential by mutations of phenylalanine-28.

Conserved amino-acids of H-ras from residues 25 to 34 were mutated in human H-ras cDNA with a pre-existing valine-12 activating mutation ([V12]p21), and built into SV40-driven expression vectors. The influence of the introduced mutations was initially screened by transfection of Rat-1 cells to score foci of transformed cells. Non-conservative mutations of amino-acids 25 (tryptophan for glutamine), 27 (asparagine for histidine) and 34 (alanine for proline) did not abrogate the transforming potential of [V12]p21. The conservative mutation of phenylalanine-28 to tryptophan ([V12W28]p21) was also still transforming. Significantly, in the absence of the valine-12 activating mutation, tryptophan-28-ras ([W28]p21) was weakly transforming while, in contrast, [V12D28]p21 was unable to transform Rat-1 cells and retarded cell growth. Analysis of the binding and dissociation of GTP and GDP to normal and mutated p21 expressed in Escherichia coli showed that [V12D28]p21 and [D28]p21 do not bind GTP. The dissociation rate of both GTP and GDP bound to [W28]p21 is increased, suggesting a mechanism for its transforming potential in Rat-1 cells. These studies illustrate the importance of phenylalanine-28 in guanine nucleotide binding by p21H-ras. The mutations described could be valuable tools in investigations of cellular signal transduction involving small GTP-binding proteins.

beta-Glucosidase activity in fetal bovine serum renders the plant glucoside, hypoxoside, cytotoxic toward B16-F10-BL-6 mouse melanoma cells.

By using p-nitrophenyl-beta-D-glucopyranoside as substrate, beta-glucosidase activity was observed in fetal bovine serum (FBS). This activity could be inhibited by heat inactivation of the serum. Gel chromatography of FBS indicated the presence of beta-glucosidase activity with an apparent molecular mass of 29 kDa. In McCoy's 5A medium supplemented with non-heat inactivated FBS, the diglucoside hypoxoside ([E]-1,5-bis[4'beta-D-glucopyranosyloxy-3'-hydroxyphenyl]pent-4-en - 1-yne) showed cytotoxicity toward B16-F10-BL-6 mouse melanoma cells. In incubations where the media were supplemented with FBS previously heat inactivated at 56 degrees C for 1 h or more, no cytotoxicity was observed in the presence of hypoxoside. The aglucone of hypoxoside, rooperol ([E]-1,5-bis[3',4'-dihydroxyphenyl]pent-4-en-1-yne), showed cytotoxicity regardless of whether the serum was heat inactivated or not. The kinetics of the heat inactivation of the beta-glucosidase activity in FBS coincided with the loss of apparent cytotoxicity of hypoxoside. High performance liquid chromatography analysis showed that rooperol could be generated by incubation of hypoxoside in non-heat inactivated FBS, but that this ability was lost in serum that was heat inactivated for 1 h or longer. Newborn bovine serum did not contain any beta-glucosidase activity whereas it was found in three different commercial sources of FBS. This observation is of practical importance because conventional heat inactivation of FBS at 56 degrees C for 30 min was not sufficient to inactivate the beta-glucosidase activity completely.

Interaction of the di-catechols rooperol and nordihydroguaiaretic acid with oxidative systems in the human blood. A structure-activity relationship.

The effects of the di-catechols rooperol [(E)-1,5-bis(3',4'- dihydroxyphenyl)pent-4-en-1-yne; P2] and nordihydroguaiaretic acid (NDGA) on oxidative systems in the human blood were investigated. P2 and NDGA gave comparable results in the inhibition of leukotriene synthesis in the polymorphonuclear leukocyte and prostaglandin synthesis in platelet microsomes. The oxidation states of myeloperoxidase in the presence of H2O2 were also similarly affected by both P2 and NDGA. In these systems, the 4'4'-beta-D-diglucopyranoside of rooperol, hypoxoside [(E)-1,5-bis(4' beta-D-glucopyranosyloxy-3'- hydroxyphenyl) pent-4-en-1-yne; P2A] had no effect. The only system which showed significant differences in the effects of the catechols was the red blood cell. NDGA in the presence of H2O2 had a pronounced haemolytic effect, which did not correlate with its ability to induce methaemoglobin formation, while P2 had a much lower haemolytic effect, but stimulated the oxidation of haemoglobin to a greater extent than NDGA. NDGA is more hydrophobic than P2 which would result in a greater membrane effect. The pent-4-en-1-yne chain which links the catechol moieties in P2 can take part in the resonance structures of the semiquinone free radical, thus assisting in its stabilization and leading to increased methaemoglobin formation. This stabilization is also demonstrated by the fact that P2A affected the oxidation of haemoglobin to nearly the same extent as NDGA.

Analysis of co-transfected expression vectors amplified by methotrexate selection in rat-1 cells.

In an earlier investigation of the influence of high level expression of p21H-ras, rat-1 cells were co-transfected with a selectable vector (pSV2Neo), an amplifiable vector (encoding dihydrofolate reductase; DHFR) and an H-ras expression vector. In this study we have analyzed the gene dose and expression levels of the three co-transfected plasmid vectors in cell lines that had been selected and isolated at different methotrexate concentrations. Growth of the cells in the absence of selection and Southern blot analyses indicate that the transfected vectors are stably co-integrated into the host genome. High expression levels from all three co-transfected vectors were evident at both the mRNA and protein levels, indicating that they are tightly linked in the host genome. The presence of a large amount of unspliced H-ras mRNA in cells expressing high levels of H-ras p21 indicates that processing of mRNA may be rate-limiting. Comparison of the gene dose and expression levels shows that the resistance of cells to increased methotrexate concentrations can occur by different mechanisms. It is concluded that co-transfection of individual plasmid vectors into rat-1 cells, followed by methotrexate selection, is an effective manner of achieving high level expression of proteins in cultured cells.

Enzymatic conversion of the major polypeptide chains of thyroglobulin.

The subunit composition of fully reduced bovine thyroglobulin consists of two polypeptide chains with molecular weights near 300,000. These two chains have been separated on sodium dodecyl sulfate gels by electrophoresis and were referred to as S and F. The effect of incubating native bovine thyroglobulin with a commercial preparation of horseradish peroxidase resulted in the conversion of S to F (and faster migrating polypeptides). Since this reaction was independent of the presence of H2O2 and iodide, it was necessary to demonstrate that the activity was present in the peroxidase enzyme which could be identified by its heme group. When the enzyme preparation was fractionated on Sephadex G-100, the activity responsible for the conversion of S to F was not associated with the heme peak but with another protein peak which eluted later on the column. Proteolytic inhibitors, such as phenylmethyl sulfonyl fluoride and pancreatic trypsin inhibitor, caused partial inhibition of the activity responsible for the conversion of S to F, whereas inhibitors of peroxidase activity had no effect. Guinea pig thyroglobulin was less susceptible to the proteolytic activity present in the peroxidase preparation than bovine thyroglobulin. The subunit composition of guinea pig thyroglobulin is different from that of bovine, since three bands of different sizes are present. The 300,000 mol wt subunit of guinea pig thyroglobulin (band A) ws largely degraded, whereas the 210,000 mol wt subunit (band B) was partially degraded and the 100,000 mol wt subunit (band C) was unaffected.