Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The genotoxic carcinogen aflatoxin B1 (AFB1) inhibited the calmodulin-stimulated membrane-bound (Ca2+Mg2+)-ATPase. Using the purified enzyme, 12 nmoles per ml of AFB1 caused maximum inhibition of 28% and 50%, of the acidic phospholipid-stimulated and calmodulin-activated Ca(2+)-ATPase activity respectively. Treatment of red cell ghosts with increasing concentrations of Triton X-100, a non-ionic detergent caused a progressive loss of both the basal and calmodulin-stimulated Ca(2+)-ATPase activity. The activity of the phospholipid-free, detergent-solubilized enzyme was almost fully restored by phosphatidyl serine (PS) and its sensitivity to calmodulin was restored in the presence of phosphatidyl choline (PC). Analysis of the results obtained using varying concentrations of ATP shows that AFB1 did not affect the Km and Vmax of the unstimulated enzyme whereas these parameters were reduced by about 75% and 50%, respectively, in the presence of calmodulin. Using the product of limited proteolysis by trypsin i.e. the 90 kDa fragment which still retains its calmodulin binding-domain and the 76 kDa fragment which has lost this domain, kinetic studies on the enzyme activity revealed that AFB1 inhibited the calmodulin-activated 90 kDa fragment by about 50% while the 76 kDa was not affected at all by the toxin and calmodulin. The toxin had no significant affect on the basal activity of the 90 kDa limited proteolysis fragment of the enzyme. These observations suggest that AFB1 inhibits the activated Ca(2+)-ATPase by binding to an important site in the calmodulin-binding domain of the enzyme. It seems likely that the toxin binds to tryptophan in the calmodulin-binding domain, thus causing a reduction in the rate at which this domain can interact with Ca(2+)-calmodulin or acidic phospholipids. The implication of these observations is that Ca(2+)-extrusion and other calmodulin-activated enzymes and processes may be slowed down during prolonged exposure to AFB1 because of its anticalmodulin effect.
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PMID:The anticalmodulin effect of aflatoxin B1 on purified erythrocyte Ca(2+)-ATPase. 856 72

Methylene blue plus visible light, in the presence of oxygen, induced lipid peroxidation in rat liver microsomes, as assessed by the formation of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides and the loss of membrane-bound enzymes. Peroxidation was enhanced by deuteration of the buffer and inhibited by scavengers of singlet oxygen (1O2) and superoxide (O2.-). The damage induced seemed to be mainly due to Type II involving 1O2 and to a lesser extent Type I reactions with O2.- and hydroxyl radical (.OH) as intermediates. Nicotinamide or vitamin B3, an endogenous metabolite occurring at high concentrations in tissues, had a relatively high rate constant of 1.8 x 108 M-1 s-1 with 102 and had a significant inhibitory effect on lipid peroxidation induced by photosensitization. This effect was both time- and concentration-dependent, high inhibition being associated with millimolar concentrations. Chemically related endogenous compounds like tryptophan and isonicotinic acid also had significant inhibitory properties. Similar protective effects were observed with natural antioxidants such as beta-carotene, canthaxanthin, lipoic acid, glutathione, alpha-tocopherol and to a lesser extent ascorbic acid. Nicotinamide was a more effective antioxidant than ascorbic acid. It also showed a similar inhibitory effect against NADPH-ADP-FE3(+)-induced lipid peroxidation. Our results suggest that nicotinamide had significant ability to protect against photosensitization-induced cytotoxicity and cell damage and that it may do so by its ability to react with 102 and other reactive oxygen species.
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PMID:Methylene blue plus light-induced lipid peroxidation in rat liver microsomes: inhibition by nicotinamide (vitamin B3) and other antioxidants. 862 May 61

A 37-residue cationic antimicrobial peptide named mesentericin Y 105(37) was purified to homogeneity from cell-free culture supernatant of the Gram-positive bacterium Leuconostoc mesenteroides. The complete amino acid sequence of the peptide, KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW, has been established by automated Edman degradation, mass spectrometry, and solid phase synthesis. Mesentericin Y 105(37) contains a single intramolecular disulfide bond that forms a 6-membered ring within the molecule. Mesentericin Y 105(37) was synthesized by the solid phase method. The synthetic replicate was shown to be indistinguishable from the natural peptide with respect to electrophoretic and chromatographic properties, mass spectrometry analysis, automated amino acid sequence determination, and antimicrobial properties. At nanomolar concentrations, synthetic mesentericin Y 105(37) is active against Gram+ bacteria in the genera Lactobacillus and Carnobacterium. Most interestingly, the peptide is inhibitory to the growth of the food-borne pathogen Listeria. CD spectra of mesentericin Y 105(37) in low polarity medium, which mimic the lipophilicity of the membrane of target organisms, indicated 30-40% alpha-helical conformation, and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning over residues 17-31. To reveal the molecular basis of the specificity of mesentericin Y 105(37) targetting and mode of action, NH2- or COOH-terminally truncated analogs together with point-substituted analogs were synthesized and evaluated for their ability to inhibit the growth of Listeria ivanovii. In sharp contrast with broad spectrum alpha-helical antimicrobial peptides from vertebrate animals, which can be shortened to 14-18 residues without deleterious effect on potency, molecular elements responsible for anti-Listeria activity of mesentericin Y 105(37) are to be traced at once to the NH2-terminal tripeptide KYY, the disulfide bridge, the putative alpha-helical domain 17-31, and the COOH-terminal tryptophan residue of the molecule. It is proposed that the amphipathic helical domain of the peptide interacts with lipid bilayers, leading subsequently to alteration of the membrane functions, whereas residues 1-14 form part of a recognition structure for a membrane-bound receptor, which may be critical for peptide targetting. Because mesentericin Y 105(37) is easy to synthesize at low cost, it may represent a useful and tractable tool as a starting point for the design of more potent analogs that may be of potential applicability in foods preservation.
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PMID:Covalent structure, synthesis, and structure-function studies of mesentericin Y 105(37), a defensive peptide from gram-positive bacteria Leuconostoc mesenteroides. 866 68

The periplasmic histidine permease of Salmonella typhimurium is composed of a membrane-bound complex and a soluble histidine-binding protein (the periplasmic receptor), HisJ. Liganded receptor interacts with the membrane-bound complex, inducing ATP hydrolysis and substrate translocation. Preliminary evidence had shown a lack of direct correlation between the affinity of HisJ for a ligand and translocation efficiency, suggesting that the precise form of the receptor is important in determining its interaction with the membrane-bound complex. We have investigated the nature of the conformations assumed by HisJ upon binding a variety of ligands by tryptophan fluorescence enhancement, reaction with a closed form-specific monoclonal antibody, and changes in UV absorption spectra. It is demonstrated that although HisJ binds all the ligands and undergoes a conformational change, it assumes measurably different conformations. We also show that the interaction between HisJ and the membrane-bound complex depends on the nature of the ligand. Transport specificity appears to be defined, at least in part, by the conformation of the bound receptor, manifested either by the effect of a given ligand on the closed structure per se, or by the effect of ligand association on the equilibrium constant relating the open and the closed liganded forms.
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PMID:Ligand-dependent conformational plasticity of the periplasmic histidine-binding protein HisJ. Involvement in transport specificity. 870 98

Systematic double-D-amino acid replacement of adjacent amino acids has been used to study the secondary structure of the amphiphilic, antibiotic peptide magainin 2 amide (M2a) by circular dichroism spectroscopy. Bound to liposomes, the secondary structure of the peptide is characterized by a weak alpha-helix in the N-terminus and a stable alpha-helix between residues 9 and 21. The lack of conformational differences in the peptide when bound to vesicles of varying negative charge density indicates marked independence of the structure from electrostatic forces. The similarity of the helicity profiles observed for double D-isomers bound to vesicles and in the presence of sodium dodecyl sulfate micelles (SDS) clearly shows that SDS can mimic magainin-lipid interactions. In contrast, in 1:1 trifluoroethanol/buffer (v/v), the peptide exhibits a weak alpha-helix extended from the N- to the C-terminus. Dye release experiments from vesicles of phosphatidylglycerol showed that double-D-amino acid substitution only in the region of the stable helix results in a reduction of the membrane-permeabilizing ability. On vesicles with a reduced amount of acidic phospholipids, double-D-amino acid substitution in any position leads to a drastic reduction of peptide-induced membrane permeabilization. Whereas the activity of M2a on phosphatidylglycerol was found to be mainly electrostatically determined, hydrophobic interactions play a decisive role in the interaction with vesicles of reduced negative charge density. Fluorescence investigations of tryptophan-containing analogs of high and low helicity showed that differences in the location of the chromophores of the membrane-bound peptides do not exist.
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PMID:Conformational and functional study of magainin 2 in model membrane environments using the new approach of systematic double-D-amino acid replacement. 871 76

Shiga toxin is a bacterial protein composed of one A and five B subunits. Its A chain possesses a protease sensitive loop (Cys-242-Cys-261) that is cleaved to produce an enzymatically active A1 domain and an A2 fragment associated with its B subunit pentamer. The proposed mode of action of the toxin is linked to its retrograde transport to the ER lumen followed by the translocation of its catalytic A1 chain to the cytoplasmic side of the ER membrane. A signal sequence-like domain (residues 220-246) which constitutes the C-terminus of the A1 chain precedes a region within the protease sensitive loop (residues 247-258) that contains known and putative cleavage sites. Two peptides corresponding to this C-terminus (residues 220-246) were chemically synthesized to investigate if this signal sequence-like domain can interact with membranes. Such a property may provide a clue to the mechanism of translocation of the A1 domain across the ER membrane. The first peptide represented the native sequence, which includes a naturally occurring cysteine at position 242 and provided a thiol moiety for the attachment of a spinlabel. A second peptide was designed to contain a single tryptophan residue (Ile232Trp) located within the hydrophobic core of the sequence which served as an intrinsic fluorescence probe. The interactions of both peptides with lipid vesicles were analyzed by circular dichroism, fluorescence, and EPR spectroscopy. The peptides lack structure in aqueous buffers and adopted an alpha-helical geometry when bound to negatively charged lipid vesicles. The addition of lipid vesicles to a solution of the tryptophan-containing peptide results in a blue shift in the wavelength of its fluorescence maxima as well as an increase in fluorescence intensity at 335 nm, suggesting that the hydrophobic core of this A1 peptide relocated to a nonpolar environment. EPR measurements of a proxyl-labeled analog of the peptide (introduced at Cys-242) indicated a decreased mobility of a fraction of the proxyl probe in the presence of lipid vesicles. At pH 7, the membrane-bound probe was completely reduced by ascorbate trapped inside vesicles but only partially reduced by ascorbate added outside the vesicles, suggesting that the C-terminal region of the peptide traversed the membrane bilayer or relocated close to the surface of its inner lipid leaflet. Finally, the peptide was shown to insert into lipid vesicles, causing the release of calcein at a high peptide:lipid ratio. These results suggest that the C-terminal tail of the A1 chain may anchor this domain into the ER membrane.
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PMID:Insertion and orientation of a synthetic peptide representing the C-terminus of the A1 domain of Shiga toxin into phospholipid membranes. 875 10

The hydropathy plot of the inwardly rectifying ROMK1 K+ channel, which reveals two transmembrane and a pore region domains, also reveals areas of intermediate hydrophobicity in the N terminus (M0) and in the C terminus (post-M2). Peptides that correspond to M0, post-M2, and a control peptide, pre-M0, were synthesized and characterized for their structure, affinity to phospholipid membranes, organizational state in membranes, and ability to self-assemble and coassemble in the membrane-bound state. CD spectroscopy revealed that both M0 and post-M2 adopt highly alpha-helical structures in 1% SDS and 40% TFE/water, whereas pre-M0 is not alpha-helical in either 1% SDS or 40% TFE/water. Binding experiments with NBD-labeled peptides demonstrated that both M0 and post-M2, but not pre-M0, bind to zwitterionic phospholipid membranes with partition coefficients of 10(3)-10(5) M-1. A surface localization for both post-M2 and M0 was indicated by NBD shift, tryptophan quenching experiments with brominated phospholipids, and enzymatic cleavage. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/ acceptor (rhodamine) peptides revealed that M0 and post-M2 can coassemble in their membrane-bound state, but cannot self-associate when membrane-bound. The results are in agreement with recent data indicating that amino acids in the carboxy terminus of inwardly rectifying K+ channels have a major role in specifying the pore properties of the channels (Taglialatela M, Wible BA, Caporaso R, Brown AM, 1994 Science 264:844-847; Pessia M, Bond CT, Kavanaugh MP, Adelman JP, 1995, Neuron 14:1039-1045). The relevance of the results presented herein to the suggested model for the structure of the ROMK1 channel and to general aspects of molecular recognition between membrane-bound polypeptides are also discussed.
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PMID:Secondary structure, membrane localization, and coassembly within phospholipid membranes of synthetic segments derived from the N- and C-termini regions of the ROMK1 K+ channel. 893 Nov 47

Conformational changes occurring upon membrane binding and subsequent insertion of staphylococcal alpha-toxin were studied using complementary spectroscopic techniques. Experimental conditions were established where binding could be uncoupled from membrane insertion but insertion and channel formation seemed to be concomitant. Binding led to changes in tertiary structure as witnessed by an increase in tryptophan fluorescence, a red shift of the tryptophan maximum emission wavelength, and a change in the near UV CD spectrum. In contrast to what was observed for the soluble form of the toxin, 78% of the tryptophan residues in the membrane-bound form were accessible to the hydrophilic quencher KI. At this stage, the tryptophan residues were not in the immediate vicinity of the lipid bilayer. Upon membrane insertion, a second conformational change occurred resulting in a dramatic drop of the near UV CD signal but an increase of the far UV signal. Tryptophan residues were no longer accessible to KI but could be quenched by brominated lipids. In the light of the available data on channel formation by alpha-toxin, our results suggest that the tryptophan residues might be dipping into the membrane in order to anchor the extramembranous part of the channel to the lipid bilayer.
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PMID:Conformational changes due to membrane binding and channel formation by staphylococcal alpha-toxin. 903 82

The effect of fatty acid binding proteins (FABPs) on two key steps of microsomal phosphatidic acid formation was examined. Rat liver microsomes were purified by size-exclusion chromatography to remove endogenous cytosolic fatty acid and fatty acyl-CoA binding proteins while recombinant FABPs were used to avoid cross-contamination with such proteins from native tissue. Neither rat liver (L-FABP) nor rat intestinal fatty acid binding protein (I-FABP) stimulated liver microsomal fatty acyl-CoA synthase. In contrast, L-FABP and I-FABP enhanced microsomal conversion of [14C]oleoyl-CoA and glycerol 3-phosphate to [14C]phosphatidic acid by 18- and 7-fold, respectively. The mechanism for this stimulation, especially by I-FABP, is not known. However, several observations presented here suggest that, like L-FABP, I-FABP may interact with fatty acyl-CoA and thereby stimulate enzyme activity. First, I-FABP decreased microsomal membrane-bound oleoyl-CoA. Second, oleoyl-CoA displaced I-FABP bound fluorescent fatty acid, cis-parinaric acid, with Ki of 5.3 microM and 1.1 sites. Third, oleoyl-CoA decreased I-FABP tryptophan fluorescence with a Kd of 4.2 microM. Fourth, oleoyl-CoA red shifted emission spectra of acrylodated I-FABP, a sensitive marker of I-FABP interactions with ligands. In summary, the results demonstrate for the first time that both L-FABP and I-FABP stimulate liver microsomal phosphatidic acid formation by enhancing synthesis of phosphatidate from fatty acyl-CoA and glycerol 3-phosphate.
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PMID:Fatty acid binding protein: stimulation of microsomal phosphatidic acid formation. 914 60

The presence of tryptophan residues as intrinsic fluorophores in most proteins makes them an obvious choice for fluorescence spectroscopic analyses of such proteins. Membrane proteins have been reported to have a significantly higher tryptophan content than soluble proteins. The role of tryptophan residues in the structure and function of membrane proteins has attracted a lot of attention. Tryptophan residues in membrane proteins and peptides are believed to be distributed asymmetrically toward the interfacial region. Tryptophan octyl ester (TOE) is an important model for membrane-bound tryptophan residues. We have characterized this molecule as a fluorescent membrane probe in terms of its ionization, partitioning, and motional characteristics in unilamellar vesicles of dioleoylphosphatidylcholine. The ionization property of this molecule in model membranes has been studied by utilizing its pH-dependent fluorescence characteristics. Analysis of pH-dependent fluorescence intensity and emission maximum shows that deprotonation of the alpha-amino group of TOE occurs with an apparent pKa of approximately 7.5 in the membrane. The fluorescence lifetime of membrane-bound TOE also shows pH dependence. The fluorescence lifetimes of TOE have been interpreted by using the rotamer model for the fluorescence decay of tryptophan. Membrane/water partition coefficients of TOE were measured in both its protonated and deprotonated forms. No appreciable difference was found in its partitioning behavior with ionization. Analysis of fluorescence polarization of TOE as a function of pH showed that there is a decrease in polarization with increasing pH, implying more rotational freedom on deprotonation. This is further supported by pH-dependent red edge excitation shift and the apparent rotational correlation time of membrane-bound TOE. TOE should prove useful in monitoring the organization and dynamics of tryptophan residues incorporated into membranes.
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PMID:Ionization, partitioning, and dynamics of tryptophan octyl ester: implications for membrane-bound tryptophan residues. 925


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