Interaction of a phosphorylated site of muscle phosphofructokinase with the activation of the enzyme by NH4+ and K+.1

The crystal structure of the cyclic peptide disulfide

has been determined by X-ray diffraction. The peptide crystallizes in the space group P2₁2₁2₁, with a = 8.646(1), b = 18.462(2), c = 19.678(3)Å and Z = 4. The molecules adopt a highly folded compact conformation, stabilized by two intramolecular 4→ 1 hydrogen bonds between the Cys (1) and Pro (2) CO groups and the Cys (4) and methylamide NH groups, respectively. The backbone conformational angles for the peptide lie very close to those expected for a 3₁₀ helix. The S-S bridge adopts a right handed twist with a dihedral angle of 82°. The structure illustrates the role of stereochemically constrained residues, in generating novel peptide conformations.     

Disulfide luminescence. Emission characteristics of cyclic tetrapeptide disulfides

Luminescence has been detected in cyclic tetrapeptide disulfides containing only nonaromatic residues. Excitation of the -S-S- n-σ transition between 280 and 290 nm leads to emission in the region 300–340 nm. The position and intensity of the emission band depends on the stereochemistry of the peptide and polarity of the solvent. Quantum yields ranging from 0.002 to 0.026 have been determined. Disulfide luminescence is quenched by oxygen and enhanced in solutions saturated with nitrogen. Contributions from disulfide linkages should be considered, when analysing the emission spectra of proteins, lacking tryptophan but having a high cystine content.     

Alamethicin and synthetic peptide fragments as uncouplers of mitochondrial oxidative phosphorylation. Effect of chain length and change

Alamethicin, its derivatives and some synthetic fragments have been shown to be uncouplers of oxidative phosphorylation in rat liver mitochondria. A minimum peptide chain length of 13 residues is necessary for this activity. Peptide esters are more efficient uncouplers than the corresponding peptide acids. Esterification of the Glu(18) γ-COOH group in alamethicin does not diminish uncoupling activity. The structural requirements for uncoupling activity parallel those determined for ionophoretic action in small, unilamellar liposomes.     

Fluorescent alamethicin fragments A study of membrane activity and aqueous phase aggregation

The linear polypeptide antibiotic alamethicin is known to form channels in artificial lipid membranes. Synthetic 13- and 17-residue alamethicin fragments, labelled with a fluorescent dansyl group at the N-terminus, have been shown to translocate divalent cations across phospholipid membranes and to uncouple oxidative phosphorylation in rat liver mitochondria, in a manner analogous to the parent peptides. From studies of the aqueous phase aggregation behavior of the peptides, as well as their interaction with rat liver mitochondria, it is concluded that the interaction of the peptides with membranes is a complex process, probably involving both aqueous and membrane phase aggregation.     

Activation of the calcium/calmodulin-dependent guanylate cyclase from Paramecium by polypeptide antibiotics and melittin

The activity of the calcium/calmodulin-regulated guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from Paramecium was stimulated by several polypeptides. The most potent activator was melittin (6-fold at 30 μM), followed by alamethicin, suzukacillin, trichotoxin and gramicidin S. Marginal effects were seen with herbicolin A and polymyxin B, whereas the following compounds had no effect: ionophore A23187, actinomycin C₁, destomycin A, gramicidin A, iturin A, nigericin, nonactin, Tü 1718B, valinomycin and synthetic peptide analogues of alamethicin. Guanylate cyclase activation was not related to ion-transport capacity or to the length of the α-helical segments. Rather, the degree of amphiphilicity seemed to be an important criterion. No difference in activation was seen between native guanylate cyclase and the reconstituted enzyme. Thus, in all likelihood, polypeptide stimulation requires the presence of the guanylate cyclase/calmodulin holo-enzyme. Guanylate cyclase activation was permanent. Enzyme kinetics, such as Michaelis-Menten behavior and non-cooperativity, were retained. Incubation with polypeptides at 37°C prior to substrate addition decreased enzyme stimulation. Activation of cGMP formation as enhanced at elevated incubation temperatures. The activation energy for hemolysis of erythrocytes favorably correlated with the extent of guanylate cyclase activation (r = 0.98), suggesting a similar mechanism of interaction with membrane constituents for both processes.     

NMR localization of the O-mycoloylation on PorH,a channel forming peptide from Corynebacterium glutamicum

PorH and PorA are two small peptides that, in complex, form a voltage-dependent ion channel in the outer membrane of Corynebacterium glutamicum. Specific post-translational modifications on PorA and PorH are required for the formation of a functional ion channel. The assignment of PorH proton NMR chemical shifts in DMSO, allowed identifying unambiguously the exact position of the PorH O-mycoloylation on Ser 56 side chain. This was further confirmed by site directed mutagenesis and mass spectrometry. Together with the previously published localization of PorA mycoloylation, this provides the complete primary structure characterization of this outer membrane porin.     

Photosynthetic activity of diimidoester-modified cells,permeaplasts, and cell-free membrane fragments of the blue-green alga Anacystis nidulans

On treating the blue-green alga Anacystis nidulans with dimethylsuberimidate up to 70% of the free NH₂ of the photosynthetic membrane is amidinated, and presumably inter- and intramolecular cross-links are established in the membrane proteins. Amidination destroys the ability of A. nidulans to photoreduce HCO₃^? but leaves the photochemical activities of Photosystems II and I nearly intact. With added electron acceptors, photosynthetic O₂ evolution can be demonstrated both with permeable cells (permeaplasts) prepared by digestion of the cell wall of dimethylsuberimidate-reacted A. nidulans with lysozyme, as well as with heavy membrane particles (36 000 × g) prepared from dimethylsuberimidate-reacted cells.Permeaplasts prepared from dimethylsuberimidate-reacted cells resist damage in hypoosmotic medium, whereas those prepared from unreacted cells are induced to release C-phycocyanin. On the other hand, the former are inactivated more easily by heat stress than the latter. On this basis, it is concluded that cross-linking with dimethylsuberimidate confers functional instability to photosynthetic membranes.     

The identification and quantification of three new 6alpha-hydroxylated corticosteroids in human neonatal urine

The 24 hours urines of six two days old fullterm newborn infants were investigated for polar corticosteroids. 6alpha-hydroxy-tetrahydrocortisone, 6alpha-hydroxy-20alpha-cortolone and 6alpha-hydroxy-20beta-cortolone were identified by gas chromatographic-mass spectrometric comparison of the urinary steroids to compounds synthesized previously. These 6alpha-hydroxylated corticosteroids as well as seven other polar corticosteroids were quantified by gas chromatography or mass fragmentography. It was shown that the newly identified steroids constituted a quantitatively important part of the neonatal urinary corticosteroids. The unconjugated- and glucuronic acid conjugated steroids were quantified separately. It was found that the extent of glucuronoconjugation decreased with increasing polarity of the steroid moiety.     

Identification of a novel bifunctional delta12/delta15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2

A new gene encoding a delta12 fatty acid desaturase-related protein was cloned from a multicellular basidiomycete Coprinus cinereus TD#822-2. The 1326 bp full-length gene, designated as Cop-odeA, codes for a putative protein of 442 amino acids with a MW of 49224. The Cop-odeA yeast transformant accumulated four new fatty acids identified as 9,12-hexadecadienoic acid, 9,12,15-hexadecatrienoic acid, linoleic acid, and alpha-linolenic acid, which comprised 8.8%, 1.0%, 29.0%, and 0.6% of the total fatty acids, respectively. The Cop-odeA protein was confirmed to be a novel bifunctional fatty acid desaturase with both high delta12 desaturase activity and unusual delta15 desaturase activity.     

A molecularly imprinted receptor for separation of testosterone and epitestosterone, based on a steroidal cross-linker

A series of molecularly imprinted polymers have been prepared and investigated as stationary phases in high performance liquid chromatography for the separation of testosterone and epitestosterone using non-polar mobile phases. The polymers were imprinted using 5α-dihydrotestosterone as template, and all retain testosterone more strongly than its 17α-OH epimer. The best polymer was prepared using trifluoromethylacrylic acid as functional monomer (interacting with the template via hydrogen bonds), divinylbenzene as ‘inert’ cross-linker, and chloroform as porogen. It also included a steroid-based cross-linker, which may interact with the template via van der Waals interactions to lend additional ‘shape selectivity’. A 250 × 4.6 mm column packed with this polymer gave baseline resolution of testosterone and epitestosterone (15 μg each) in under 20 min. Preparation of the steroid based cross-linker included the selective reduction of 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) to the 3α,17β-diol using K-selectride.     

Crosstalk between Na+,K+-ATPase and a volume-regulated anion channel in membrane microdomains of human cancer cells

Low concentrations of cardiac glycosides including ouabain, digoxin, and digitoxin block cancer cell growth without affecting Na⁺,K⁺-ATPase activity, but the mechanism underlying this anti-cancer effect is not fully understood. Volume-regulated anion channel (VRAC) plays an important role in cell death signaling pathway in addition to its fundamental role in the cell volume maintenance. Here, we report cardiac glycosides-induced signaling pathway mediated by the crosstalk between Na⁺,K⁺-ATPase and VRAC in human cancer cells. Submicromolar concentrations of ouabain enhanced VRAC currents concomitantly with a deceleration of cancer cell proliferation. The effects of ouabain were abrogated by a specific inhibitor of VRAC (DCPIB) and knockdown of an essential component of VRAC (LRRC8A), and they were also attenuated by the disruption of membrane microdomains or the inhibition of NADPH oxidase. Digoxin and digitoxin also showed anti-proliferative effects in cancer cells at their therapeutic concentration ranges, and these effects were blocked by DCPIB. In membrane microdomains of cancer cells, LRRC8A was found to be co-immunoprecipitated with Na⁺,K⁺-ATPase α1-isoform. These ouabain-induced effects were not observed in non-cancer cells. Therefore, cardiac glycosides were considered to interact with Na⁺,K⁺-ATPase to stimulate the production of reactive oxygen species, and they also apparently activated VRAC within membrane microdomains, thus producing anti-proliferative effects.     

Backbone dynamics of alamethicin bound to lipid membranes: spin-echo electron paramagnetic resonance of TOAC-spin labels

Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and that induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogs in which a selected Aib residue (at position n = 1, 8, or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in association with phosphatidylcholine bilayer membranes. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2τ. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.     

Structure of Self-Aggregated Alamethicin in ePC Membranes Detected by Pulsed Electron-Electron Double Resonance and Electron Spin Echo Envelope Modulation Spectroscopies

PELDOR spectroscopy was exploited to study the self-assembled super-structure of the [Glu(OMe)^7,18,19]alamethicin molecules in vesicular membranes at peptide to lipid molar ratios in the range of 1:70-1:200. The peptide molecules were site-specifically labeled with TOAC electron spins. From the magnetic dipole-dipole interaction between the nitroxides of the monolabeled constituents and the PELDOR decay patterns measured at 77 K, intermolecular-distance distribution functions were obtained and the number of aggregated molecules (n ≈ 4) was estimated. The distance distribution functions exhibit a similar maximum at 2.3 nm. In contrast to Alm16, for Alm1 and Alm8 additional maxima were recorded at 3.2 and ∼5.2 nm. From ESEEM experiments and based on the membrane polarity profiles, the penetration depths of the different spin-labeled positions into the membrane were qualitatively estimated. It was found that the water accessibility of the spin-labels follows the order TOAC-1 > TOAC-8 ≈ TOAC-16. The geometric data obtained are discussed in terms of a penknife molecular model. At least two peptide chains are aligned parallel and eight ester groups of the polar Glu(OMe)^18,19 residues are suggested to stabilize the self-aggregate superstructure.     

Lipidomics reveals membrane lipid remodelling and release of potential lipid mediators during early stress responses in a murine melanoma cell line

Membranes are known to respond rapidly to various environmental perturbations by changing their composition and microdomain organization. In previous work we showed that a membrane fluidizer benzyl alcohol (BA) could mimic the effects of heat stress and enhance heat shock protein synthesis in different mammalian cells. Here we explore heat- and BA-induced stress further by characterizing stress-induced membrane lipid changes in mouse melanoma B16 cells. Lipidomic fingerprints revealed that membrane stress achieved either by heat or BA resulted in pronounced and highly specific alterations in lipid metabolism. The loss in polyenes with the concomitant increase in saturated lipid species was shown to be a consequence of the activation of phopholipases (mainly phopholipase A₂ and C). A phospholipase C–diacylglycerol lipase–monoacylglycerol lipase pathway was identified in B16 cells and contributed significantly to the production of several lipid mediators upon stress including the potent heat shock modulator, arachidonic acid. The accumulation of cholesterol, ceramide and saturated phosphoglyceride species with raft-forming properties observed upon both heat and BA treatments of B16 cells may explain the condensation of ordered plasma membrane domains previously detected by fluorescence microscopy and may serve as a signalling platform in stress responses or as a primary defence mechanism against the noxious effects of stresses.     

Partial site-specific assignment of a uniformly C, N enriched membrane protein, light-harvesting complex 1 (LH1), by solid state NMR

Partial site-specific assignments are reported for the solid state NMR spectra of light-harvesting complex 1, a 160 kDa integral membrane protein. The assignments were derived from 600 MHz ¹⁵N-¹³CO-¹³Cα and ¹⁵N-¹³Cα-¹³CX correlation spectra, using uniformly ¹³C, ¹⁵N enriched hydrated material, in an intact and precipitated form. Sequential assignments were verified using characteristic ¹⁵N-¹³Cα-¹³Cβ side chain chemical shifts observed in 3D experiments. Tertiary contacts found in 2D DARR spectra of the selectively ¹³C enriched sample provided further confirmatory evidence for the assignments. The assignments include the region of the Histidine ligands binding the Bacteriochlorophyll chromophore. The chemical shifts of Cα and Cβ resonances indicated the presence of typical α-helical secondary structure, consistent with previous studies.     

TOAC spin labels in the backbone of alamethicin: EPR studies in lipid membranes

Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe)(7,18,19) variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC(n) alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic (14)N-hyperfine couplings indicate that TOAC(8) and TOAC(16) are situated in the hydrophobic core of the membrane, whereas the TOAC(1) label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.     

Effects of agronomic application of olive mill wastewater in a field of olive trees on carbohydrate profiles, chlorophyll a fluorescence and mineral nutrient content

Olive mill wastewater (OMW) management is a serious environmental issue for the Mediterranean area where there is the most production of olive oil. OMW contains a high organic load, substantial amounts of plant nutrients but also several compounds with recognized toxicity towards living organisms. Moreover, OMW may represent a low cost source of water. We studied the influence of irrigation with OMW (amounts applied: 30, 60, 100 and 150 m³ h⁻¹) in a field of olive trees on root colonization, photosynthesis, chlorophyll fluorescence, leaf nutrient concentration and soluble carbohydrate. The soil fatty acid methyl ester (FAME) 16:1ω5 was used to quantify biomass of arbuscular mycorrhizal (AM) fungi and the root FAME 16:1ω5 analysis was used as index for the development of colonization in the roots. Agronomic application of OMW decreased significantly the abundance of the soil FAME 16:1ω5 and the root FAME 16:1ω5 in the soil amended with 60, 100 and 150 m³ ha⁻¹ OMW. Decreased root FAME 16:1ω5 due to OMW amendment was associated with a significant reduction of tissue nutrient concentrations in the olive trees. The highest application of OMW to the soil reduced significantly the olive trees uptake of N, P, K, Ca, Mg, Fe, Cu, Mn and Zn. Land spreading of OMW increased concentration of soluble carbohydrate in the olive leaves, mostly due to decreased sink demand for carbon by the root. In the olive trees amended with 150 m³ ha⁻¹ OMW, net CO₂ uptake rate (A), quantum yield of photosystem II electron transport (Φ_PSII), maximal photochemical efficiency of photosystem II (Fv/Fm), photochemical quenching (q_p) and the electron transport rate (ETR) were significantly depressed, whereas non-photochemical quenching (NPQ) was found to increase. Taken with data from experiments in field conditions, our results suggest that agronomic application of OMW alters the functioning of arbuscular mycorrhizas and can even disrupt the relationship between AM fungi and olive trees.     

Lipid chain-length dependence for incorporation of alamethicin in membranes: electron paramagnetic resonance studies on TOAC-spin labeled analogs

Alamethicin is a 19-residue hydrophobic peptide, which is extended by a C-terminal phenylalaninol but lacks residues that might anchor the ends of the peptide at the lipid-water interface. Voltage-dependent ion channels formed by alamethicin depend strongly in their characteristics on chain length of the host lipid membranes. EPR spectroscopy is used to investigate the dependence on lipid chain length of the incorporation of spin-labeled alamethicin in phosphatidylcholine bilayer membranes. The spin-label amino acid TOAC is substituted at residue positions n = 1, 8, or 16 in the sequence of alamethicin F50/5 [TOAC(n), Glu(OMe)(7,18,19)]. Polarity-dependent isotropic hyperfine couplings of the three TOAC derivatives indicate that alamethicin assumes approximately the same location, relative to the membrane midplane, in fluid diC(N)PtdCho bilayers with chain lengths ranging from N = 10-18. Residue TOAC(8) is situated closest to the bilayer midplane, whereas TOAC(16) is located farther from the midplane in the hydrophobic core of the opposing lipid leaflet, and TOAC(1) remains in the lipid polar headgroup region. Orientational order parameters indicate that the tilt of alamethicin relative to the membrane normal is relatively small, even at high temperatures in the fluid phase, and increases rather slowly with decreasing chain length (from 13 degrees to 23 degrees for N = 18 and 10, respectively, at 75 degrees C). This is insufficient for alamethicin to achieve hydrophobic matching. Alamethicin differs in its mode of incorporation from other helical peptides for which transmembrane orientation has been determined as a function of lipid chain length.