New fluorescent octadecapentaenoic acids as probes of lipid membranes and protein-lipid interactions.

The chemical and spectroscopic properties of the new fluorescent acids all(E)-8, 10, 12, 14, 16-octadecapentaenoic acid (t-COPA) and its (8Z)-isomer (c-COPA) have been characterized in solvents of different polarity, synthetic lipid bilayers, and lipid/protein systems. These compounds are reasonably photostable in solution, present an intense UV absorption band (epsilon(350 nm) approximately 10(5) M(-1) cm(-1)) strongly overlapped by tryptophan fluorescence and their emission, centered at 470 nm, is strongly polarized (r(O) = 0.385 +/- 0.005) and decays with a major component (85%) of lifetime 23 ns and a faster minor one of lifetime 2 ns (D,L-alpha-dimyristoylphosphatidylcholine (DMPC), 15 degrees C). Both COPA isomers incorporate readily into vesicles and membranes (K(p) approximately 10(6)) and align parallel to the lipids. t-COPA distributes homogeneously between gel and fluid lipid domains and the changes in polarization accurately reflect the lipid T(m) values. From the decay of the fluorescence anisotropy in spherical bilayers of DMPC and POPC it is shown that t-COPA also correctly reflects the lipid order parameters, determined by 2H NMR techniques. Resonance energy transfer from tryptophan to the bound pentaenoic acid in serum albumin in solution, and from the tryptophan residues of gramicidin in lipid bilayers also containing the pentaenoic acid, show that this probe is a useful acceptor of protein tryptophan excitation, with R(O) values of 30-34 A.     

Lipid-protein interactions in rat renal subcellular membranes: a biophysical and biochemical study

The phase behavior of plasma membrane (PM), endoplasmic reticulum (ER), and nuclear membranes (NM) isolated from adult rat papillary cells was studied using the molecular probe Laurdan. The steady-state fluorescence data analysis was correlated with the lipid composition obtained by biochemical assays. The comparison between intact membranes and protein-free reconstituted vesicles using the whole lipid extract shows the essential role of proteins on the temperature response of natural membranes. The phospholipid (PL) and cholesterol (Cho) content was measured in the three membrane fractions, the PL/Cho molar ratio being between 1.5 and 1.9. However, Laurdan's parameters in NM show a fluid phase state pattern even at low temperature (5 degrees C), with a restricted dipole relaxation in comparison with that displayed in liquid crystalline phase state lipid model membranes. PM and ER are in a gel-like state at temperatures below 20 degrees C, showing increasing dipole relaxation with temperature. The curved fits obtained are characteristic of cholesterol-enriched membranes. The distinctive phase behavior of nuclear membranes vanishes when proteins are extracted. However, relaxation is still faster in this fraction, which correlates with the native lipid composition. NM has the lowest percentage of phosphatidylinositol and sphingomyelin-the latter being a highly saturated phospholipid- and the highest percentage of phosphatidylcholine and phosphatidylethanolamine (PE), nuclear PE being enriched in arachidonic acid. All these changes agree with the higher fluidity of NM compared with ER or PM in the conditions assayed.     

Thiophosphorylation as a probe for subunit interactions in Escherichia coli succinyl coenzyme A synthetase. Further evidence for catalytic cooperativity and substrate synergism

Succinyl-CoA synthetase has an (alpha beta)2 subunit structure and shows half-of-the-sites reactivity with respect to the formation of the phosphohistidyl residues that acts as a catalytic intermediate. Adenosine 5'-O-(3-thio)triphosphate has been found to be a substrate, but the overall maximum velocity is 3 orders of magnitude lower than that seen with ATP. Moreover, steps of the reaction involving thiophosphoryl transfer are much slower than the corresponding phosphoryl transfers. These properties of adenosine 5'-O-(3-thio)triphosphate as a substrate have been exploited to test the concept of alternating sites catalytic cooperativity proposed earlier as a rationale for the subunit structure of succinyl-CoA synthetase. As predicted by this model for catalysis, the rate of discharge of thiophosphate from the enzyme in the presence of succinate and CoA is stimulated by ATP. Neither of two nonhydrolyzable analogs of ATP has an equivalent effect. The results indicate that the transfer of the thiophosphoryl group from the enzyme to succinate at one active site is not favored until the neighboring active site is phosphorylated by ATP, with accompanying reciprocal changes in the conformations of the two halves of the enzyme molecule.     

The reactivity of one essential cysteine as a conformational probe in Escherichia coli tryptophanase. Application to the study of the structural influence of subunit interactions.

It is first shown that the inactivation of Escherichia coli apotryptophanase byN-ethylmaleimide results from the labeling of a single particularly reactive cysteine per protomer. The reactivity of this cysteine under various conditions is investigated and the results indicate that the protein can exist under two classes of conformation: one, corresponding to inactive protein,in which the cysteine is reactive, and second, corresponding to active enzyme, where the cysteine is masked. The rate of the isomeriation step involved in this change in conformation is measured by the stopped-flow technique(tou = 0.4s). Finally, the reactivity of the cysteine is used to characterize the conformation of dimeric holotryptophanase (i.e. a dissociated form of the enzyme obtained as a transient species between dimeric apoenzyme and the natural tetrameric holoenzyme). By this criterion, it is shown that dimeric holotryptophanase falls in the class of 'inactive' conformations. These results are used to discuss the influence of the quaternary structure on the functional and conformational properties of tryptophanase and the nature of the conformational change involved in the activation of the enzyme by its cofactor and specific cations.     

Incorporation of diaminopimelic acid into Escherichia coli membranes

Abstract Pulse-labeled, cell division-inhibited Escherichia coli incorporate [³H]diamino pimelic acid (DAP) into membrane fractions of light (= inner membrane) and intermediate densities and transfer the DAP rapidly into heavy membrane fractions (= outer membrane). The intermediate density fraction which has been shown to contain markers for membrane adhesion sites appears to serve as intermediary in the translocation.     

Fluorescence study of protein-lipid complexes with a new symmetric squarylium probe

The novel symmetric squarylium derivative SQ-1 has been synthesized and tested for its sensitivity to the formation of protein-lipid complexes. SQ-1 binding to the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in different molar ratios was found to be controlled mainly by hydrophobic interactions. Lysozyme (Lz) and ribonuclease A (RNase) exerted an influence on the probe association with lipid vesicles resulting presumably from the competition between SQ-1 and the proteins for bilayer free volume and modification of its properties. The magnitude of this effect was much higher for lysozyme which may stem from the amphipathy of protein alpha-helix involved in the membrane binding. Varying membrane composition provides evidence for the dye sensitivity to both hydrophobic and electrostatic protein-lipid interactions. Fluorescence anisotropy studies uncovered the restriction of SQ-1 rotational mobility in lipid environment in the presence of Lz and RNase being indicative of the incorporation of the proteins into bilayer interior. The results of binding, fluorescence quenching and kinetic experiments suggested lysozyme-induced local lipid demixing upon protein association with negatively charged membranes with threshold concentration of CL for the lipid demixing being 10 mol%.     

A new mercury-penicillin V derivative as a probe for ultrastructural localization of penicillin-binding proteins in Escherichia coli.

The precise ultrastructural localization of penicillin-binding protein (PBP)-antibiotic complexes in Escherichia coli JM101, JM101 (pBS96), and JM101(pPH116) was investigated by high-resolution electron microscopy. We used mercury-penicillin V (Hg-pen V) as a heavy-metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells grown to exponential growth phase. Biochemical data derived from susceptibility tests and bacteriolysis experiments revealed no significant differences between Hg-pen V and the parent compound, penicillin V, or between strains. Both antibiotics revealed differences in the binding affinities for PBPs of all strains. Deacylation rates for PBPs were slow despite the relatively low binding affinities of antibiotics. Cells bound most of the Hg-pen V added to cultures, and the antibiotic-PBP complex could readily be seen by electron microscopy of unstained whole mounts as distinct, randomly situated electron-dense particles. Fifty to 60% of the antibiotic was retained by cells during processing for conventional embedding so that thin sections could also be examined. These revealed similar electron-dense particles located predominantly on the plasma membrane and less frequently in the cytoplasm. Particles positioned on the plasma membranes were occasionally shown to protrude into the periplasmic space, thereby reflecting the high resolution of the Hg-pen V probe. Moreover, some particles were observed free in the periplasm, suggesting, for the first time, that a proportion of PBPs may not be restricted to the plasma membrane but may be tightly associated with the peptidoglycan for higher efficiency of peptidoglycan assembly. All controls were devoid of the electron-dense particles. The presence of electron-dense particles in cells of the wild-type JM101, demonstrated that our probe could identify PBPs in naturally occurring strains without inducing PBP overproduction.     

Thermodynamic interactions of a cis and trans benzanilide with Escherichia coli bacterial membranes

The activity of a membrane interactive cis and trans benzanilide against Escherichia coli membrane mimics was investigated using Langmuir monolayers. It was found that in the presence of E. coli lipid mix monolayers, cis-benzanilide induced maximal surface pressure changes of 1?mN?m(-1), whereas a reduced interaction was observed with trans-benzanilide. Compression isotherm analysis of these monolayers showed ?G (mix)?相似文献   

Calcium and chlorpromazine interactions in rat synaptic plasma membranes. A spin-label and fluorescence probe study.

The effects of calcium and of the psychoactive drug chlorpromazine (CPZ) on the rat synaptic plasma membrane have been studied using two stearic nitroxide spin labels having their doxyl groups in positions 5 and 16 and the fluorescent probe 1-anilinonaphtalene-8-sulfonate (ANS). The mobility of the 5-doxyl stearic spin label which probes the membrane phospholipids in the vicinity of their polar heads is decreased in the presence of both compounds. Calcium is more efficient in this respect than CPZ. In spite of this qualitative similarity of action, CPZ inhibits the effect of calcium and vice versa. No modification of the 16-doxyl stearic spectrum has been observed even at high calcium or CPZ concentrations. An increase in fluorescence intensity and a blue shift in the emission wavelength of ANS-probed membranes are observed with very low CPZ concentrations (10^?7 to 10^?5m). With higher concentrations, a further intensity increase and a further blue shift are due to direct interaction between ANS and CPZ. Calcium also increases the fluorescence intensity of ANS-labeled membranes in the concentration range 10^?5–10^?2m. As for the spin-label data, the effects of both compounds are mutually competitive. It is concluded that calcium interacts principally with the phospholipid polar heads of this type of membrane. However, the competition with CPZ suggests indirectly that this ion is also bound to membrane proteins. CPZ has an affinity for membrane lipids only at high concentrations. In its pharmacologically active concentration range, it is located preferentially on the membrane proteins.     

Accumulation of nucleotides by starved Escherichia coli cells as a probe for the involvement of ribonucleases in ribonucleic acid degradation.

The acid-soluble ribonucleic acid degradation products formed by Escherichia coli cells starved for a carbon source have been identified. They comprise oligonucleotides, nucleoside diphosphates, 5'- and 3'-nucleoside monophosphates, nucleosides, and free bases. The majority of these products are excreted phates, nucleosides, and free bases. The majority of these products are excreted into the medium, and only small and constant amounts are kept in the pool. During carbon starvation at elevated temperatures, mutants deficient in ribonuclease I do not form oligonucleotides and 3'-nucleoside monophosphates, and mutants that contain a modified form of polynucleotide phosphorylase do not accumulate nucleoside diphosphates. 5'-Nucleoside monophosphates do accumulate, however, in a mutant containing thermoabile ribonuclease II, under conditions where more than 95% of all enzyme activity had been destroyed. The data presented confirm the participation of ribonuclease I and polynucleotide phosphorylase in the final steps of ribonucleic acid degradation and indicate that an exonuclease forming 5'-nucleoside monophosphates is also involved.     

Lipid dynamics and protein-lipid interactions in rat colonic epithelial cell basolateral membranes

Lipid dynamics and lipid-protein interactions were examined in basolateral membranes prepared from rat proximal and distal colonic epithelial cells. The results demonstrate that: (1) these membranes have a high lipid fluidity, as assessed by steady-state fluorescence polarization studies using seven fluorescent probes; (2) lipid compositional differences exist between these membranes but their fluidity is similar; (3) fluorescence polarizations studies, using diphenylhexatriene (DPH), detect a thermotropic transition at 22–23°C in each membrane; (4) several membrane protein activities, including adenylate cyclase and sodium-potassium dependent adenosine triphosphatase ($\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$) appear to be functionally dependent on the physical state of the proximal basolateral membrane's lipid.     

Modification of the biophysical characteristics of membranes in commensal Escherichia coli strains from breast cancer patients

Escherichia coli G35 N49 strain, from the gut of breast cancer patients, in comparison with the E. coli G35 N61 strain, from the gut of healthy people, shows in vitro reduction in growth rates and maximal growth yield. The changes in certain membrane characteristics, such as low membrane potential and disturbance in intramembrane interaction of H+ -ATPase F0F1 with the TrkA system, indicate a dysfunction in ion transport and enzymatic activity. These changes can be detected during fermentation and in anaerobic conditions (in the gut, for example) and may be influenced by unfavorable conditions in the gut of cancer patients.     

Pyrenesulfonyl azide as a fluorescent label for the study of protein-lipid boundaries of acetylcholine receptors in membranes

Acetylcholine receptor (AcChR) enriched membrane fragments from Torpedo californica electroplax were labeled by in situ photogenerated nitrenes from a hydrophobic fluorescent probe, pyrene-1-sulfonyl azide. Preferential photolabeling of membrane proteins, mainly AcChR, has been achieved and there is a pronounced exposure of the 48,000 and 55,000 molecular weight subunits of AcChR to the lipid environment of the membrane core. Covalent attachment of the photogenerated fluorescence probe does not perturb the α-neurotoxins' binding properties of membrane-bound AcChR or the desensitization kinetics induced by prolonged exposures to cholinergic agonists. Non-covalent photoproducts can be conveniently removed from labeled membrane preparations by exchange into lipid vesicles prepared from electroplax membrane lipids. Fluorescence features of model pyrene sulfonyl amide derivatives, such as fine vibrational structure of emission spectra or fluorescence lifetimes, are highly sensitive to the solvent milieu. The covalently bound probe shows similar fluorescence properties in situ. PySA photoproducts have great potential to spectroscopically monitor neurotransmitter induced events on selected AcChR subunits exposed to the hydrophobic environment of membranes.     

Compartmentation of gramicidin 5 in membranes of sensitive bacteria and protein-lipid interactions]

Gramicidin S is sorbed on the isolated membranes of granicidin-sensitive Micrococcus lysodeikticus strain. The antibiotic inhibits the membrane malate dehydrogenase within the temperature range of 9--42 degrees C, i.e. under conditions of gel and liquid-crystalline lipid state; however its effect at 10 degrees C is 10 times as low as is observed at 42 degrees C. The inhibitory effect of gramicidin S on malate dehydrogenase can be eliminated and the antibiotic can be removed from the membrane by an excess of different phospholipids. No transfer of the membrane components on exogenous phospholipids is observed. A prolonged (about 2 hrs, 30 degrees C) incubation of the membranes with gramicidin S results in irreversible inactivation of malate dehydrogenase, although the antibiotic can be still eliminated by an addition of phospholipid emulsions. It is suggested that gramicidin S forms complexes with phospholipids, in which the antibiotic is oriented to water. These complexes disturb the lipid-protein interactions, resulting in relaxation of the binding between the boundary phospholipids and proteins, in the loosening of near-protein lipid zones and simultaneous condensation of acid phospholipids in the whole membrane. Destruction of the lipid zone is accompanied by changes in the enzyme activity, by separation of lipid and protein regions and by transphase enzyme transitions (expulsion or immersion). A slow formation of secondary protein-protein associates may be irreversible.     

Interaction of Escherichia coli hemolysin with biological membranes. A study using cysteine scanning mutagenesis.

Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins. Lytic activity depends on binding of Ca2(+) to the C-terminus of the molecule. The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain. In this study, 13 HlyA cysteine-replacement mutants were constructed and labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane-bound toxin. Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca2(+), suggesting that Ca2(+) is not required for the toxin to enter membranes. However, binding of Ca2(+) to HlyA in solution effected conformational changes in both the C-terminal and N-terminal domain that paralleled activation. Our data indicate that binding of Ca2(+) to the toxin in solution effects a conformational change that is relayed to the N-terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.