Focused proteomics: monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain

We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue.     

Crystallization of chicken liver (basic) fatty acid binding protein after purification in multicompartment electrolyzers with isoelectric membranes

A preparation of chicken liver (basic) fatty acid binding protein was purified to homogeneity in multicompartment electrolyzers with isoelectric membranes. Large amounts of the isoelectric point (pI) 9.7 protein were collected into a compartment delimited by pI 8.8 and 11.0 membranes. The protein thus purified produced crystals which diffract to higher resolution than those obtained by purification via preparative isoelectric focusing (IEF) in soluble carrier ampholytes. In addition, a novel orthorhombic form with a different molecular packing was obtained. It is hypothesized that, when using conventional IEF, traces of carrier ampholytes could adhere to the protein, particularly in the hydrophobic ligand-binding pocket, rendering the interpretation of the electron density maps difficult. Multicompartment electrolyzers do not present this drawback, since they are based on insoluble buffering species.     

Potential of peptides selected from random phage-displayed libraries to mimic conformational epitopes: a study on scorpion toxin Cn2 and the neutralizing monoclonal antibody BCF2

Many conformational epitopes cannot be mapped by the use of a phage display approach due to the lack of amino acid similarity with the selected peptides. Exploring the potential of the method, we selected mimotopes of the discontinuous, highly conformational epitope of scorpion neurotoxin Cn2, whose 3D structure is known, using its generic neutralizing monoclonal antibody BCF2. With an exhaustive selection procedure, we isolated from a 12-mer phage library a large collection of mimotopes that reproduce the antigenic and immunogenic specificity of the Cn2-epitope. The selected peptides presented three sequence motifs, the most abundant of which, RD(N)XXGF, appeared in 15 different sequence contexts displayed by 97 out of 206 clones. In the most reactive mimotope, displayed by 24 (25%) clones, the motif was flanked by two Cys residues allowing the adoption of a cyclic conformation. Motifs QL(H,M)L(M) and (S/T)WHLP were selected with less efficiency. Comparison of the motifs with the primary and three-dimensional structure of Cn2 as well as with a model of the Cn2-BCF2(Fv) complex suggests that RD(N)XXGF, which does not share sequence similarity with the epitope, mimics its central structural element, turn 7-11, by using an alternative amino acid combination nevertheless keeping the nature of its interactions with BCF2. The QL(H,M)L(M) is assumed to mimic the hydrophobic part of the epitope. The principles of the conformational mimicry by phage-displayed peptides are discussed.     

Isolation of the alanine carrier from the membranes of a thermophilic bacterium and its reconstitution into vesicles capable of transport.

A carrier protein mediating alanine transport was purified from the membranes of the thermophilic bacterium PS3, by ion exchange chromatography in the presence of both Triton X-100 and urea. The alanine carrier was recovered in the nonadsorbed fraction from either DEAE- or CM-cellulose columns, suggesting that its isoelectric point was in the neutral pH region. The final preparation contained virtually no electron transfer components, ATPase, or NADH dehydrogenase. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the final preparation consisted of two major protein components with molecular weights of 36,000 and 9,400. Active transport of alanine after incorporation of the alanine carrier into reconstituted proteoliposomes was driven not only by an artificial membrane potential generated by potassium ion diffusion via valinomycin but also by mitochondrial cytochrome oxidase incorporated into the same liposomes and supplemented with both cytochrome c and ascorbic acid. The membrane-integrated portion (TFo) of the ATPase complex uncoupled alanine transport by conducting protons across the membrane.     

Recognition based separation of HIV-Tat protein using avidin–biotin interaction in modified microfiltration membranes

Recognition based separation using modified microfiltration membranes provides an efficient and cost-effective alternative to conventional column chromatography for the separation and purification of a specific protein from mixture of proteins. In this study, Tat protein, which has been proposed as the specific target for AIDS vaccine, was separated and purified from a complex mixture of proteins, known as bacterial lysate (BL) using avidin–biotin interaction in 4-stack microfiltration membranes system. It was established by SDS-PAGE and Western Blot analysis that membrane based process recovered more pure form of Tat compared to conventional packed-bead column chromatography. The critical factors involved in the process, mainly, the accessibility of the covalently immobilized avidin sites by the biotinylated protein and the associated fouling of the membranes due to the permeation of proteins, were also studied. The accessibility of immobilized avidin sites in membrane was quantified by biotinylated solutions of different types and compositions. It was observed that permeation of proteins caused substantial fouling on the membrane matrix. The resistance offered by the protein layer and the approximate thickness of the protein layer were also quantified.     

Purification and characterization of a major glycoprotein in rat liver lysosomal membrane

A major lysosomal membrane glycoprotein (LGP107) which has an apparent molecular weight (Mr) of 107 kilodaltons (kDa) was purified from rat liver by a simple method with a yield of 1 mg/87 g wet weight of liver. The purification procedures include; preparation of tritosomal membranes of triton-filled lysosomes (tritosomes), extraction of tritosomal membranes by Lubrol PX, wheat germ agglutinin (WGA)-Sepharose affinity chromatography, and monoclonal antibody-Sepharose affinity chromatography. The quantitative immunoblot analysis indicated that LGP107 represents 6.2% of the total protein of tritosomal membranes. The isoelectric point of the purified glycoprotein was 2.7, and it moved toward neutral pH after sialidase treatment, with its molecular weight decreased by about 10 kDa. LGP107 contained 52% carbohydrates, and the carbohydrate moiety was compared of Fuc, Man, Gal, GlcNAc and sialic acid in a molar ratio of 7.2:68.2:40.6:63.0:32.3, respectively, indicating that LGP107 was highly glycosylated with N-linked complex-type olgosaccharide chains. Out of the N-linked glycans released from the glycoprotein by hydrazinolysis/N-reacetylation, about 70% was sialylated. Anion exchange and reverse-phase high performance liquid chromatography analysis on the structure of N-glycans revealed that a disialyl biantennary form is a major component in the oligosaccharide chains of LGP107.     

Partial purification of active delta and epsilon subunits of the membrane ATPase from escherichia coli.

We have partially purified active delta and epsilon subunits of the E. coli membrane-bound Mg2+-ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (alpha, beta, and gamma) of the enzyme, but the two minor subunits (delta and epsilon), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five-subunit ATPase and the enzyme deficient in the delta subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two-subunit enzyme, consisting chiefly of the alpha and beta subunits, was insensitive to the ATPase inhibitor, suggesting that the gamma subunit may be required for inhibition by epsilon. The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase-depleted membranes and to reconstitute ATP-dependent transhydrogenase. Previously we reported (Biochem, Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1.     

Orientation-selective incorporation of transmembrane F0F1 ATP synthase complex from micrococcus luteus in polymer-supported membranes

We report the vectorial incorporation of a highly asymmetric F0F1 ATP synthase complex from Micrococcus luteus into polymer-supported membranes. Dynamic light scattering and cryo electron microscopy confirm that the use of weak surfactants (bile acid) allows for the non-disruptive protein incorporation into lipid vesicles. Spreading of vesicles with ATP synthase onto a cellulose support results in a homogeneous distribution of proteins, in contrast to a patchy image observed on bare glass slides. The orientation of ATP synthase can be identified using an antibody to the ATP binding site as well as from topographic profiles of the surface. The method to "align" transmembrane proteins in supported membranes would open a possibility to quantify protein functions in biomimetic model systems.     

Phosphotyrosine phosphatases acting on band 3 in human erythrocytes of different age: PTP1B processing during cell ageing

The phosphorylation of tyrosine residues of human red blood cell (RBC) band 3 is regulated in vivo by constitutively active tyrosine-kinases (PTKs) and phosphotyrosine-phosphatases (PTPs), identified so far as, respectively, p72(syk) and p56/53(lyn), and PTP1B and SHPTP-2. Tyr-phosphorylation of band 3 increases upon reduction of cell volume as in hypertonic media or during Ca(2+)-induced membrane vesiculation. We show here that old RBCs display higher Tyr-phosphorylation levels of band 3 than younger cells under hypertonic conditions, at least in part due to the reduced cell volume of old RBCs, a condition of lowered threshold for activation of volume-sensitive PTKs. We have also analysed the membrane-bound PTP activity and the relative abundance of PTP1B (as the main membrane-associated PTP) in RBCs of different age. Immunodetection of PTP1B in purified ghost membranes revealed that the catalytic, N-terminal domain of the PTP is partially cleaved, in an age-dependent manner, from the membrane-bound domain, and it is lost during the preparation of ghost membranes. This suggests that erythrocytes may undergo in vivo activation of the Ca(2+)-dependent calpain system that proteolytically regulates PTP1B activity, as already documented for other cell types. On the other hand, the assay of the PTP activity that remains associated with the membranes of RBCs of different age indicated that the PTP undergoes oxidative inactivation that can be further differentiated into reversible and irreversible components.     

Synthesis and Crystal Structure of ［Na（DB18C6）（CH_3OH）］_2Mo_6O_（19）·（DB18C6）·（CH_3OH）

ＳｙｎｔｈｅｓｉｓａｎｄＣｒｙｓｔａｌＳｔｒｕｃｔｕｒｅｏｆ［Ｎａ（ＤＢ１８Ｃ６）（ＣＨ_３ＯＨ）］_２Ｍｏ_６Ｏ_（１９）·（ＤＢ１８Ｃ６）·（ＣＨ_３ＯＨ）￥ＺｈｏｕＹｉｎ－Ｚｈｕａｎｇ；ＴｕＳｈｕ－Ｊｉｅ；ＪｉｎＸｉａｎｇ－Ｌｉｎ；ＬｉｕＳｈｕｎ－...     

Probing molecular-level surface structures of polyethersulfone/pluronic F127 blends using sum-frequency generation vibrational spectroscopy

We blended Pluronic F127 into polyethersulfone (PES) to improve surface properties of PES, which has been extensively used in biomaterial and other applications. The molecular surface structures of PES/Pluronic F127 blends have been investigated by sum-frequency generation (SFG) vibrational spectroscopy. The molecular orientation of surface functional groups of PES changed significantly when blended with a small amount of Pluornic F127. Pluronic F127 on the blend surface also exhibited different features upon contacting with water. The entanglement of PES chains with Pluronic F127 molecules rendered the blends with long-term surface stability in water in contrast to the situation where a layer of Pluronic F127 adsorbed on the PES surface. Atomic force microscopy (AFM) and quartz crystal microbalance (QCM) measurements were included to determine the relative amount of protein that adsorbed to the blend surfaces. The results showed a decreased protein adsorption amount with increasing Pluronic F127 bulk concentration. The correlations between polymer surface properties and detailed molecular structures obtained by SFG would provide insight into the designing and developing of biomedical polymers and functional membranes with improved fouling-resistant properties.     

Chromatographic methods for large-scale preparation of immunoglobulin G2a monoclonal antibodies Lym-1 and TNT-1 F(ab')2 fragments

Lym-1 and TNT-1 are two murine immunoglobulin G2a monoclonal antibodies (MAbs) which have been used for clinical trials in cancer patients. This paper describes methods for large-scale preparation of F(ab')2 fragments from 50 mg to 4 g of MAbs Lym-1 and TNT-1. Digestion of MAbs with pepsin was optimized and performed at pH 3.8, a pepsin/antibody ratio of 1:250, and 3-4 h of incubation at 37 degrees C. The F(ab')2 fragments were purified by tandem column procedures using fast protein liquid chromatography. Quality control analyses of the products included protein purity, isoelectric point, immunoreactivity, and endotoxin level. The results revealed that the chromatographic procedures are practical, simple, and effective, and can be used to produce gram quantities of clinical-grade F(ab')2 fragments for the diagnosis of cancer in patients.     

Remarkable reduction of irreversible fouling and improvement of the permeation properties of poly(ether sulfone) ultrafiltration membranes by blending with pluronic F127

Hydrophilic modification of ultrafiltration membranes was achieved through blending of Pluronic F127 with poly(ether sulfone) (PES). The chemical composition and morphology changes of the membrane surface were confirmed by water contact angle, X-ray photoelectron spectroscopy, scanning electron microscopy, and protein adsorption measurements. The decreased static water contact angle with an increase in the Pluronic F127 content indicated an increase of surface hydrophilicity. XPS analysis revealed enrichment of PEO segments of Pluronic F127 at the membrane surface. The apparent protein adsorption amount decreased significantly from 56.2 to 0 microg/cm(2) when the Pluronic F127 content varied from 0% to 10.5%, which indicated that the blend membrane had an excellent ability to resist protein adsorption. The ultrafiltration experiments revealed that the Pluronic F127 content had little influence on the protein rejection ratio and pure water flux. Most importantly, at a high Pluronic F127 content membrane fouling, especially irreversible fouling, has been remarkably reduced. The flux recoveries of blend membranes reached as high as 90% after periodic cleaning in three cycles.     

Crystal Structure of Binuclear Bridging Complex[(dppf)_2Pd_2 (Ⅱ) (μ-Cl)_2] [PF_6]_2

1INTRODUCTIoNFerrocenecontainingtransitionmeta1complexeshasarousedtheinterestofchemistsbecauseofitsnovelstructureandproperties"',Huangetal[2'3isynthesizedsomechainandbridgeformcomplexesandstudiedtheirIRandCV.Linetal[4isyn-thesizedthecrystalof[(dppf)PdCl,jO.5[(CH3),COj(dppf=1,11-(diphenylphosphine)-ferrocene)anddeterminedthestructurebyX-raydiffraction.Inthisarticlewereportthesynthesisandcrystalstructureof[(dppf),Pd,(ll)(p-Cl),j[PF,j2.2EXPERIMENTAL2.1PreparationAsolutionofO.86mm…     

Small-angle X-ray scattering study of photosystem I-detergent complexes: implications for membrane protein crystallization

Small-angle X-ray scattering (SAXS) was used to investigate the structure of isolated photosystem I (PSI) complexes stabilized in detergent solution. Two different types of PSI preparation were investigated. In the first preparation, thylakoid membranes were solubilized with Triton X100 and purified by density gradient centrifugation. SAXS data indicated large scattering objects or microphases that can be described as sheets with approximately 68 A thickness and a virtually infinite lateral extension. The observed thickness agreed well with the dimension of a PSI molecule across the thylakoid membrane. In the second preparation, PSI was isolated as before but was further purified by anion exchange chromatography resulting in functional complexes consisting of single PSI units with attached surfactant as evidenced by the particle volume and gyration radius extracted from the SAXS data. Several approaches were used to model the solution conformation of the complex. Three different ellipsoidal modeling approaches, a uniform density ellipsoid of revolution, a triaxial solid ellipsoid, and a core-shell model, found extended structures with dimensions that were not consistent with the PSI crystal structure (Ben-Shem, A.; et al. Nature 2003, 426, 630-635). Additionally, the SAXS data could not be modeled using the crystal structure embedded in a disk of detergent. The final approach considered the possibility that protein was partially unfolded by the detergent. The data were modeled using a "beads-on-a-string" approach that describes detergent micelles associated with the unfolded polypeptide chains. This model reproduced the position and relative amplitude of a peak present in the SAXS data at 0.16 A(-1) but was not consistent with the data at larger length scales. We conclude that the polypeptide subunits at the periphery of the PSI complex were partially unfolded and associated with detergent micelles while the catalytically active core of the PSI complex remained structurally intact. This interpretation of the solution structure of isolated PSI complexes has broader implications for the investigation of the interactions of detergents and protein, especially for crystallization studies.     

Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin

The release of neurotransmitter is regulated in the processes of membrane docking and membrane fusion between synaptic vesicles and presynaptic plasma membranes. Synaptic vesicles contain a diverse set of proteins that participate in these processes. Small GTP-binding proteins exist in the synaptic vesicles and are suggested to play roles for the regulation of neurotransmitter release. We have examined a possible role of GTP-binding proteins in the regulation of protein phosphorylation in the synaptic vesicles. GTPgammaS stimulated the phosphorylation of 46 kDa protein (p46) with pI value of 5.0-5.2, but GDPbetaS did not. The p46 was identified as protein interacting with C-kinase 1 (PICK-1) by MALDI-TOF mass spectroscopy analysis, and anti-PICK-1 antibody recognized the p46 spot on 2-dimensional gel electrophoresis. Rab guanine nucleotide dissociation inhibitor (RabGDI), which dissociates Rab proteins from SVs, did not affect phosphorylation of p46. Ca(2+)/calmodulin (CaM), which causes the small GTP-binding proteins like Rab3A and RalA to dissociate from the membranes and stimulates CaM-dependent protein kinase(s) and phosphatase, strongly stimulate the phosphorylation of p46 in the presence of cyclosporin A and cyclophylin. However, RhoGDI, which dissociates Rho proteins from membranes, reduced the phosphorylation of p46 to the extent of about 50%. These results support that p46 was PICK-1, and its phosphorylation was stimulated by GTP and Ca(2+)/CaM directly or indirectly through GTP-binding protein(s) and Ca(2+)/CaM effector protein(s). The phosphorylation of p46 (PICK-1) by GTP and Ca(2+)/CaM may be important for the regulation of transporters and neurosecretion.     

Specific Lipid Studies in Complex Membranes by Solid-State NMR Spectroscopy

Specific interactions with phospholipids are often critical for the function of proteins or drugs, but studying these interactions at high resolution remains difficult, especially in complex membranes that mimic biological conditions. In principle, molecular interactions with phospholipids could be directly probed by solid-state NMR (ssNMR). However, due to the challenge to detect specific lipids in mixed liposomes and limited spectral sensitivity, ssNMR studies of specific lipids in complex membranes are scarce. Here, by using purified biological ¹³C,¹⁵N-labeled phospholipids, we show that we can selectively detect traces of specific lipids in complex membranes. In combination with ¹H-detected ssNMR, we show that our approach provides unprecedented high-resolution insights into the mechanisms of drugs that target specific lipids. This broadly applicable approach opens new opportunities for the molecular characterization of specific lipid interactions with proteins or drugs in complex fluid membranes.     

Turmeric Extract (Curcuma longa) Mediates Anti-Oxidative Effects by Reduction of Nitric Oxide,iNOS Protein-, and mRNA-Synthesis in BV2 Microglial Cells

Plant-derived products have been used since the beginnings of human history to treat various pathological conditions. Practical experience as well as a growing body of research suggests the benefits of the use of turmeric (Curcuma longa) and some of its active components in the reduction of oxidative stress, a mechanism leading to neurodegeneration. In this current study, we investigated the effects of a preparation of Curcuma longa, and its constituents curcumin, tetrahydrocurcumin, and curcumenol, in one of the molecular pathways leading to oxidative stress, which is the release of NO, a free radical involved in stress conditions, using the BV2 microglial cell line. The concentration-dependent reduction of NO is linked to reduced amounts of iNOS protein- and mRNA-synthesis and is possibly mediated by the phosphorylation of mitogen-activated protein kinases (MAPK) such as p42/44 or p38 MAPK. Therefore, the use of turmeric extract is a promising therapeutic option for diseases linked to the dysregulation of oxidative stress, with fewer side-effects in comparison to the currently used pharmacotherapeutics.     

Synthesis and Crystal Structure of Binuclear Oxomolybdenum(VI) Complex with Both o-Mercaptophenolate

1INTRoDUCTIONMolybdenumintheVandVIoxidationstatesformsaconsiderablenumberofoxocomplexes.Someofthemareactivecatalystsfortheoxidationoforganicsub-stracts[li'Allthesefactshavewidelyarousedchemists'interest.lnordertoobservetheeffectofthedonorgroupsonthedegreeofcomplexaggregationandonthecoor-dinationgeometry,thetitlecompoundwaspreparedbythereactionof(n-Bu'N)'-[Mo,O,,jwitho-mercaptophenolandthesinglecrystalswereobtained.Itcontainsa[Mo,O,j' core.Somereleventreportscanbefoundinthepreviousliter…     

Phospholipase D is involved in oxidative stress-induced migration of vascular smooth muscle cells via tyrosine phosphorylation and protein kinase C

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H(2)O(2) induced tyrosine phosphorylation of PLD1, protein kinase C-alpha (PKC-alpha), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-alpha in VSMCs. Stimulation of the cells by H(2)O(2) and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H(2)O(2)-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H(2)O(2)-stimulated PLD activation. The cells stimulated by oxidative stress (H(2)O(2)) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.