Identification of a novel 300-kDa factor termed IkappaB alphaE3-F1 that is required for ubiquitinylation of IkappaB alpha. (25/1497)

Destruction of IkappaB by ubiquitinylation is required for signal-dependent activation of NF-kappaB. The IkappaB alpha ubiquitin-ligase activity associated with phosphorylated IkappaB alpha (pIkappaB alpha) in HeLa cells was almost completely lost by washing under stringent conditions including 1 M NaCl; nevertheless, an SCF(betaTrCP) complex containing Skp1, Cullin-1, and F-box/WD40 protein betaTrCP was still bound to pIkappaB alpha, suggesting the existence of a putative factor that is loosely associated with pIkappaB alpha and may collaborate with SCF(betaTrCP). The factor was named IkappaB alphaE3-F1 and was partially purified from HeLa cells. Gel filtration analysis revealed that IkappaB alphaE3-F1 has an apparent molecular mass of approximately 300 kDa.  (+info)

The chimeric peptide [Lys(-2)-Arg(-1)]-sarafotoxin-S6b, composed of the endothelin pro-sequence and sarafotoxin, retains the salt-bridge staple between Arg(-1) and Asp8 previously observed in [Lys(-2)-Arg(-1)]-endothelin. Implications of this salt-bridge in the contractile activity and the oxidative folding reaction. (26/1497)

The chimeric peptide [Lys(-2)-Arg(-1)]-sarafotoxin-S6b (KR-SRTb) designed from the Lys-2-Arg-1 dipeptide of the endothelin pro-sequence and the sarafotoxin-S6b sequence was synthesized. Its contractile activity was found to be decreased markedly when compared with that of the parent SRTb. In contrast, the extension by the Lys-Arg dipeptide was found to increase the formation of the native disulfide isomer (82/18 versus 96/4) when the reaction was carried out in the presence of redox reagents. The solution structure of KR-SRTb was determined by NMR as a function of pH. In the carboxylic acid state, the structure consists of the cystine-stabilized alpha-helical motif, with the alpha-helical part spanning residues 9-15, and of an unstructured C-terminal tail. In the carboxylate state, the structure is characterized by a salt-bridge between Arg(-1) and Asp8, which we identified previously in the [Lys(-2)-Arg(-1)]-endothelin-1 peptide (KR-ET-1). The fact that this salt-bridge is commonly observed in KR-SRTb and KR-ET-1, despite the 33% sequence difference between the corresponding parental peptides, highlights the remarkable adaptability of the Lys-Arg extension for the formation of a special salt-bridge. As a consequence, this salt-bridge, which does not depend on either the 4-7 sequence of the loop or the C-terminal sequence, appears to be particularly well suited to improve the stability of the cystine-stabilized alpha-helical motif. Therefore, because of its high yield in the native disulfide arrangement and its high permissiveness for sequence mutation in the 4-7 loop, such a stabilized cystine-stabilized alpha-helical motif could be a valuable scaffold for the presentation of a library of constrained short peptides.  (+info)

The major cell surface glycoprotein of chick embryo fibroblasts is an agglutinin. (27/1497)

A major cell surface protein, CSP, of chick embryo fibroblasts has been shown to constitute up to 3% of total cell protein, and to be decreased after viral transformation. Its role in normal cell behavior is not known. We have isolated CSP from chick embryo fibroblasts by extraction with 1 M urea and find that these preparations of CSP agglutinate formalinized sheep erythrocytes at protein concentrations of under 2 mug/ml. In extracts of chick embryo cells, the quantity of such hemagglutinating activity parallels that of CSP determined by electrophoresis, and both are substantially decreased in chick cells transformed by the Bryan hightiter strain of Rous sarcoma virus. Both CSP and hemagglutinating activity are progressively adsorbed onto erythrocytes and can be released by 1 M urea. An antiserum to purified CSP specifically blocks the agglutination. The agglutinating activity is destroyed by boiling or treatment with proteases. The agglutination reaction is inhibited by the chelating agents EDTA and EGTA [ethyleneglycol-bis(beta-aminoethyl ether)N,N'-tetraacetic acid]. Agglutination is also inhibited to a lesser degres by amino sugars and other amines, increased osmolarity, and urea. Other monosaccharides, hyaluronidase, DNase, and RNase have little or not effect on the agglutination reaction. This demonstration that CSP has an agglutinating activity that is sensitive to proteases and that requires divalent cations suggests that this molecule may play a role in cell adhesion.  (+info)

Extracellular nuclease from Rhizopus stolonifer: purification and characteristics of - single strand preferential - deoxyribonuclease activity. (28/1497)

An extracellular nuclease from Rhizopus stolonifer (designated as nuclease Rsn) was purified to homogeneity by chromatography on DEAE-cellulose followed by Blue Sepharose. The M(r) of the purified enzyme determined by native PAGE was 67 inverted question mark omitted inverted question mark000 and it is a tetramer and each protomer consists of two unidentical subunits of M(r) 21 inverted question mark omitted inverted question mark000 and 13 inverted question mark omitted inverted question mark000. It is an acidic protein with a pI of 4.2 and is not a glycoprotein. The purified enzyme showed an obligate requirement of divalent cations like Mg(2+), Mn(2+) and Co(2+) for its activity but is not a metalloprotein. The optimum pH of the enzyme was 7.0 and was not influenced by the type of metal ion used. Although, the optimum temperature of the enzyme for single stranded (ss) DNA hydrolysis in presence of all three metal ions and for double stranded (ds) DNA hydrolysis in presence of Mg(2+) was 40 degrees C, it showed higher optimum temperature (45 degrees C) for dsDNA hydrolysis in presence of Mn(2+) and Co(2+). Nuclease Rsn was inhibited by divalent cations like Zn(2+), Cu(2+) and Hg(2+), inorganic phosphate and pyrophosphate, low concentrations of SDS, guanidine hydrochloride and urea, organic solvents like dimethyl sulphoxide, dimethyl formamide and formamide but not by 3'- or 5'-mononucleotides. The studies on mode and mechanism of action showed that nuclease Rsn is an endonuclease and cleaves dsDNA through a single hit mechanism. The end products of both ssDNA and dsDNA hydrolysis were predominantly oligonucleotides ending in 3'-hydroxyl and 5'-phosphoryl termini. Moreover, the type of metal ion used did not influence the mode and mechanism of action of the enzyme.  (+info)

Influence of a lysine 331 counterion on the pK(a) of aspartic acid 125: evidence for a salt-bridge interaction and role in alpha(1b)-adrenergic receptor activation. (29/1497)

We have hypothesized previously that a salt-bridge constraint exists in the alpha(1b)-adrenergic receptor (AR). Docking of the agonist epinephrine can disrupt this constraint via competition of its protonated amine, leading to an agonist-induced activation of second messengers. The amino acids, K331 and D125, which comprise this salt-bridge, should be closely associated with each other in the unbound form of the alpha(1b)-AR. This ionic association should stabilize the negative charge of D125, leading to an increase in its acid strength or a decreased pK(a). If the charged state of D125 is important for agonist binding, then changing the type of amino acid at position 331 should decrease the acid strength of D125, leading to epinephrine affinity changes for the alpha(1b)-AR. To test this hypothesis, site-directed mutagenesis was performed at position 331 of the alpha(1b)-AR. The effect these substitutions had on D125 acid strength was quantitated via epinephrine affinity changes calculated from competition binding experiments performed at different pH values. For all mutations of the alpha(1b)-AR where the positive charge at position 331 was eliminated, there was a significant increase in the pK(a) ( congruent with 0.73) of an acidic amino acid(s). In addition, there was an increase in the binding affinity of epinephrine for these mutants that was associated with a gain in the basal production of inositol triphosphates. These results are consistent with an aspartic acid residue as the counterion for K331 of the salt-bridge constraint, which disrupted, is a part of the receptor activation process. Moreover, changes in the pK(a) of D125 were not dependent on the type of amino acid substituted at position 331. This suggests a mechanism in which K331 is no longer influencing D125 after salt-bridge disruption in the wild-type alpha(1b)-AR, but may move to another stabilized position, analogous to what has been suggested for bacteriorhodopsin. Differences from the wild-type receptor in D125 pK(a) for the K331 mutations were used to estimate the free-energy potential of the constraining salt-bridge. This free energy ( congruent with 1 kcal/mol) is significant, but weak enough to be consistent with an activational mechanism where docking of the receptor agonist has sufficient free energy to cause disruption of the salt-bridge.  (+info)

The delivery of salts to the xylem. Three types of anion conductance in the plasmalemma of the xylem parenchyma of roots of barley. (30/1497)

To explore possible pathways for anions to enter the xylem in the root during the transport of salts to the shoot, we used the patch-clamp method on protoplasts prepared from the xylem parenchyma of barley (Hordeum vulgare L.) plants. K(+) currents were suppressed by tetraethylammonium or N-methylglucamine in the solutions in the pipette and the bath, and the permeating anions were Cl(-) or NO(3)(-). We recorded the activities of three distinct anion conductances: (a) an inwardly rectifying anion channel (X-IRAC), characterized by activation at hyperpolarization and open times of up to several seconds; (b) a quickly activating anion conductance (X-QUAC), important for anion efflux at voltages between -50 mV and the equilibrium potential of the prevailing anion; and (c) a slowly activating anion conductance (X-SLAC), activating above -100 mV. Both X-IRAC and X-QUAC were permeable for Cl(-) and NO(3)(-); X-QUAC was also permeable for malate. The occurrence of X-IRAC became more frequent with an increase in cytoplasmic Ca(2+), while the occurrence of X-QUAC decreased. Anion currents through X-SLAC, and particularly through X-QUAC, were estimated to be large enough to account for reported rates of xylem loading, which is in accordance with the notion that xylem loading is a passive process.  (+info)

Structural and kinetic studies of lasalocid A (X537A) and its silver, sodium, and barium salts in nonpolar solvents. (31/1497)

The ionophore lasalocid A (X537A) and its metal salts have been investigated by high resolution (270 MHz and 360 MHz) proton nuclear magnetic resonance spectroscopy to obtain structural and kinetic information in nonpolar solution. The proton resonances were assigned from double resonance studies on lasalocid A and on its salts, homologs, isomers, and chemically modified derivatives. Studies of proton and carbon longitudinal relaxation time suggest that lasalocid A exists as a monomer, whereas the sodium and barium salts exist as dimers in nonpolar solvents. A study of the magnitude of the vicinal proton coupling constants and the chemical shifts and linewidths of the hydroxyl resonances suggest that the backbone conformation and intramolecular hydrogen bonds are similar for lasalocid A and its sodium and barium salts in nonpolar solvents. Nuclear magnetic resonance studies on the role of bound solvent molecules suggest a tightly bound water molecule in the barium complex dimer (crystallized from water-ethanol) and a weakly bound ethanol molecule in the lasalocid A monomer (crystallized from ethanol) in cyclohexane. The selective changes in proton chemical shift on complexation [where the polar faces of two lasalocid anions coordinate the metal cation(s) in nonpolar solvents have been analyzed in terms of the proximity of the resonances to the cation, their linkage to the coordinating oxygen atoms, and the magnetic anisotropy effects of the polar groups of one ligand on the resonances of its partner in the dimer. The nuclear magnetic resonance studies in solution are compared with earlier observations on lasalocid A and its salts in the crystalline state. Thus, the short Ag-C5 distance in the crystal structure of silver complex dimer is also observed in the solution structure. The kinetic parameters associated with the exchange between lasalocid A and its barium complex in chloroform have been measured from an analysis of the resonance line shapes as a function of temperature.  (+info)

Identification of the site where the electron transfer chain of plant mitochondria is stimulated by electrostatic charge screening. (32/1497)

Modular kinetic analysis was used to determine the sites in plant mitochondria where charge-screening stimulates the rate of electron transfer from external NAD(P)H to oxygen. In mitochondria isolated from potato (Solanum tuberosum L.) tuber callus, stimulation of the rate of oxygen uptake was accompanied by a decrease in the steady-state reduction level of coenzyme Q, and by a small decrease in the steady-state reduction level of cytochrome c. Modular kinetic analysis around coenzyme Q revealed that stimulation of the rate was due to stimulation of quinol oxidation via the cytochrome pathway (cytochrome bc1, cytochrome c and cytochrome c oxidase). It was not a consequence of any effect on quinone reduction (by external NADH or NADPH dehydrogenase). This explains the salt-induced decrease in the steady-state reduction level of coenzyme Q. Analysis around cytochrome c revealed that stimulation by salts was due to a dual effect on the respiratory chain. The kinetic curves for the oxidation and reduction pathways of cytochrome c revealed that they were both activated by salt, the simultaneity explaining the small variation observed in the steady-state reduction level of cytochrome c. A simple kinetic core model is used to show that changes in the rate of dissociation of cytochrome c from the membrane can explain the observed kinetic changes in both cytochrome c reduction and cytochrome c oxidation. The stimulation is proposed to be the result of an increase in the rate constant of cytochrome c dissociation from the membrane induced by cation screening. We conclude that this type of modular kinetic analysis is a powerful tool to identify and quantitatively characterize multiple-site effects on the mitochondrial respiratory chain.  (+info)