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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The affinity between CrIII and purified calf- thymus DNA was studied at neutral pH by competition with ethidium bromide. Competition results indicated an affinity between CrIII and DNA of the order of 10(5) 1/mole. These results are in good agreement with previous results CrIII - DNA affinity was studied by the independent method of equilibrium dialysis and chromium dosage by atomic spectrometry.
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PMID:[Affinity between CrIII and purified DNA, studied by competition with an intercalating agent: ethidium bromide]. 729

The 7-ethoxycoumarin O-deethylase activity of rat cytochrome P450 (P450) 2B1 was inactivated by 9-ethynylphenanthrene (9EPh) in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. At 20 degrees C, the extrapolated maximal rate constant of inactivation (kinactivation) was 0.45 min-1 and the inactivator concentration required for half-maximal inactivation (KI) was 138 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC analysis demonstrated that [2'-3H]-9EPh was irreversibly bound to the protein moiety of P450 2B1 and the stoichiometry of binding was determined to be 0.82 mol of inactivator bound per mole of P450 2B1. A radiolabeled peptide of approximately 3.0 kDa was identified by autoradiography after Tricine SDS-PAGE analysis of the peptides generated from a cyanogen bromide cleavage of [2'-3H]9EPh-inactivated P450 2B1. After HPLC separation of these peptides, the fraction containing the most radioactivity was analyzed by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) and peaks at m/z 2720.9 and 2939.9 were detected. The lower mass peak represents the molecular ion (MH+) for the peptide Ile290 to Met314 (theoretical 2722.2), while the higher mass peak corresponds to the MH+ of the modified peptide (theoretical 2940.5). The difference in mass (approximately 219) would correspond to the addition of a phenanthrylacetyl group to the peptide. When the fraction containing the modified and unmodified peptides was further digested with pepsin and reanalyzed by MALDI-MS, the site of attachment could be assigned to one of the amino acids contained in the peptide Phe297 to Leu307.
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PMID:Mechanism-based inactivation of cytochrome P450 2B1 by 9-ethynylphenanthrene. 748 91

Anion-selective channels from apical membranes of cultured CFPAC-1 cells were isolated and incorporated into giant liposomes for patch clamp recording. Liposomes were formed from L-alpha-lecithin by a dehydration-hydration method. Ion channels were characterized using the excised inside-out patch clamp configuration. The most commonly observed anion channels were similar to those observed in native epithelial tissues. The linear 20 pS Cl- channel had the halide permeability sequence Cl- > I- > or = Br- > F-, and showed anomalous mole-fraction behavior in solutions containing different proportions of Cl- and F- ions. The autwardly rectifying Cl- channel had the halide permeability sequence I- > Br- > Cl- > F-, and also showed anomalous mole-fraction behavior, indicating that both these channels probably contain multi-ion pores. The third, voltage-dependent anion channel showed at least five different substrates, had a conductance of 390 pS in the main state, and showed two types of kinetics, fast (openings and closings < 1 ms), and slow (openings and closings > 1 s). The channel was seen more frequently after reconstitution into giant liposomes than in intact cells. It was not selective amongst the halides, and there was no deviation from a linear dependence of relative current on molar fractions, indicating relatively simple permeation through the pore. Differences in halide permeabilities suggest that different anion channels may be related to different membrane proteins. Comparison with the chloride channel proteins isolated biochemically from epithelial cell membranes is discussed.
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PMID:Halide permeation through three types of epithelial anion channels after reconstitution into giant liposomes. 768 90

Single-channel currents through GABA- and glycine-activated chloride channels of post-natal tissue-cultured hippocampal neurons were measured to determine their anion selectivity and their concentration dependence of permeation. Current-voltage relations for both agonists displayed rectification with single-channel conductance increasing at positive potentials. Permeabilities determined from reversal potentials were maximal for anions with a diameter of about 4 A. Larger diameter anions had lower permeabilities, consistent with an approximate pore diameter of 6 A for both agonist-activated channels. The permeability for anions of similar size was greatest for those ions with a more symmetrical charge distribution (e.g. NO3- > Bicarbonate-). The permeability sequence was SCN- > NO3- > I- > Br- > Cl- > Formate- > Acetate- > Bicarbonate- > Gluconate- > F- > Phosphate-, whereas the conductance sequence for anion efflux was Cl- > Br- > NO3- > I- > SCN- > Formate- > Acetate- > Bicarbonate- > Gluconate- > F- > Phosphate-. These results suggest that the ions interact with sites within the channel, with hydration forces contributing an important component to the barrier for ion entry into the channel. The spherically symmetrical halides displayed an exponential relation between relative permeability and hydration energy. Concentration dependence of conductance for Cl- channels in symmetrical Cl- solutions with agonist in the pipette showed an increase at positive potentials and a decrease at negative potentials. GABA- and glycine-activated channels also exhibited anomalous mole-fraction effects in a mixture of Cl- and SCN-. These results suggest that both agonist-activated channels act as multi-ion pathways and have similar permeation characteristics.
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PMID:Anion permeation in GABA- and glycine-gated channels of mammalian cultured hippocampal neurons. 769 Apr 84

Staphylococcus hyicus lipase is a serine hydrolase. In order to identify the active site histidine of S. hyicus lipase we have chemically modified S. hyicus lipase with 1-bromo-octan-2-one. The enzyme is rapidly inactivated by this inhibitor with a half-time of 578 s at pH 6.5 and 30 degrees C. Addition of the enzyme's cofactor calcium increases the inactivation rate approx. 2-fold. When n-hexadecylphosphocholine, a non-hydrolysable substrate analogue, is added the inactivation rate decreases about 3-fold, suggesting that a residue in the active site of S. hyicus lipase is involved in the inactivation reaction. Inactivation of S. hyicus lipase with 14C-labelled 1-bromo-octan-2-one shows that 1.4 moles of inhibitor per mole of lipase are incorporated. The results of an electrospray mass spectrometric study of the inactivated enzyme are consistent with this finding. In order to identify the modified residue, both the inactivated and the unmodified lipase were digested with cyanogen bromide followed by trypsin. The resulting peptides were analysed using HPLC and fast atom bombardment mass spectrometry. The results allow the modified residue to be assigned to the peptide Gly597-Lys612. Collision induced dissociation mass spectrometry allowed the modified residue to be identified as His-600. From these results we conclude that this residue forms part of the catalytic triad of S. hyicus lipase.
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PMID:Identification of the active site histidine in Staphylococcus hyicus lipase using chemical modification and mass spectrometry. 771 Oct 54

Native calponin is able to bind 2 mol of calcium binding protein (CaBP) per mole calponin. This study extends this observation to define the 2 domains of interaction, one of which is near the actin binding site, and the other in the amino-terminal region of calponin. Also, the first evidence for a differentiation in the response of calponin to interaction with caltropin versus calmodulin is demonstrated. The binding of caltropin to cleavage and recombinant fragments of calponin was determined by 3 techniques: tryptophan fluorescence of the fragments, CD measurements to determine secondary structure changes, and analytical ultracentrifugation. In order to delineate the sites of interaction, 3 fragments of calponin have been studied. From a cyanogen bromide cleavage of calponin, residues 2-51 were isolated. This fragment is shown to bind to CaBPs and the affinity for caltropin is slightly higher than that for calmodulin. A carboxyl-terminal truncated mutant of calponin comprising residues 1-228 (CP 1-228) has been produced by recombinant techniques. Analytical ultracentrifugation has shown that CP 1-228, like the parent calponin, is able to bind 2 mol of caltropin per mol of 1-228 in a Ca(2+)-dependent fashion, indicating that there is a second site of interaction between residues 52-228. Temperature denaturation of the carboxyl-terminal truncated fragment compared with whole calponin show that the carboxyl-terminal region does not change the temperature at which calponin melts; however, there is greater residual secondary structure with whole calponin versus the fragment. A second mutant produced through recombinant techniques comprises residues 45-228 and is also able to bind caltropin, thus mapping the location of the second site of interaction to near the actin binding site.
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PMID:Two domains of interaction with calcium binding proteins can be mapped using fragments of calponin. 775 87

In a series of related papers, we have recently presented the results of a thermodynamic approach to the conformational transitions of bulk chromatin induced in vitro by different structure-perturbing agents, such as the intercalating dye ethidium bromide or the ionic strength. In all these studies, we took advantage of the capability of differential scanning calorimetry to detect the changes in the melting behavior of the structural domains of chromatin (the linker and the core particle) associated with the order-disorder transitions. This technique also revealed that the higher-order structure undergoes a catastrophic decondensation process in the course of the transformation of rat hepatocytes as well as of cultured cells. Therefore, several questions arose concerning the biological function (if any) of the changes in the degree of condensation of bulk chromatin, as well as the mechanism of transition and the nature of the modulating agents. In this paper, we report a thermodynamic analysis of the reconstitution of H1-depleted calf thymus chromatin with the purpose of establishing (1) the binding mode of H1 and (2) the energetics and cooperativity of the transition from the unfolded to the condensed state. When H1 is progressively extracted from calf thymus nuclei by high-salt treatment, the endotherm at 107 degrees C, characteristic of the core particles interacting within condensed domains, converts into the thermal transition at 90 degrees C, resulting from the denaturation of noninteracting core particles. Binding of H1 fully restores the thermal profile of native chromatin. Analysis of H1 association shows that binding occurs at independent sites with KA = (3.67 +/- 0.60) x 10(4) M-1 and each site comprising 180 +/- 10 bp. The experimental dependence of the fraction of condensed chromatin on R, the moles of bound H1 per nucleosome mole, was compared with a simple thermodynamic model for the conformational change. This analysis yields a value of -5 kcal per nucleosome mole for the interaction free energy of nucleosomes within the ordered state. The process of condensation, is not, however, a highly cooperative (all-or-none) one, as expected from a consideration of the solenoidal model for the 30 nm fiber. Rather, nucleation of the helical state involves the face-to-face interaction between consecutive core particles, and the growth is largely determined by the mergence and rearrangement of neighboring clusters of helically arrayed nucleosomes.
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PMID:Role of H1 in chromatin folding. A thermodynamic study of chromatin reconstitution by differential scanning calorimetry. 781 11

Myeloperoxidase and eosinophil peroxidase catalyzed the oxidation of bromide ion by hydrogen peroxide (H2O2) and produced a brominating agent that reacted with amine compounds to form bromamines, which are long-lived oxidants containing covalent nitrogen-bromine bonds. Results were consistent with oxidation of bromide to an equilibrium mixture of hypobromous acid (HOBr) and hypobromite ion (OBr-). Up to 1 mol of bromamine was produced per mole of H2O2, indicating that bromamine formation prevented the reduction of HOBr/OBr- by H2O2 and the loss of oxidizing and brominating activity. Bromamines differed from HOBr/OBr- in that bromamines reacted slowly with H2O2, were not reduced by dimethyl sulfoxide, and had absorption spectra similar to those of chloramines, but shifted 36 nm toward higher wavelengths. Mono- and di-bromo derivatives (RNHBr and RNHBr2) of the beta-amino acid taurine were relatively stable with half-lives of 70 and 16 h at pH 7, 37 degrees C. The mono-bromamine was obtained with a 200-fold excess of amine over the amount of HOBr/OBr- and the di-bromamine at a 2:1 ratio of HOBr/OBr- to the amine. In the presence of physiologic levels of both bromide (0.1 mM) and chloride (0.1 M), myeloperoxidase and eosinophil peroxidase produced mixtures of bromamines and chloramines containing 6 +/- 4% and 88 +/- 4% bromamine. In contrast, only the mono-chloramine derivative (RNHCl) was formed when a mixture of hypochlorous acid (HOCl) and hypochlorite ion (OCl-) was added to solutions containing bromide and excess amine. The rapid formation of the chloramine prevented the oxidation of bromide by HOCl/OCl-, and the chloramine did not react with bromide within 1 h at 37 degrees C. The results indicate that when enzyme-catalyzed bromide or chloride oxidation took place in the presence of an amine compound at 10 mM or higher, bromamines were not produced in secondary reactions such as the oxidation of bromide by HOCl/OCl- and the exchange of bromide with chlorine atoms of chloramines. Therefore, the amount of bromamine produced by myeloperoxidase or eosinophil peroxidase was equal to the amount of bromide oxidized by the enzyme. Bromide was preferred over chloride as the substrate for both enzymes.
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PMID:Oxidation of bromide by the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase. Formation of bromamines. 785 68

A Ni(2+)-binding protein (pNiXc, 40 kDa), present in Xenopus laevis oocytes and embryos, was isolated from mature oocytes by chromatography on DEAE-cellulose and cellulose phosphate, followed by FPLC on Ni-iminodiacetate-Agarose, or reverse-phase HPLC on a C-4 column. Size-exclusion HPLC showed that intact pNiXc is approximately 155 kDa, consistent with tetrameric structure. After cleavage with Lys-C proteinase or cyanogen bromide, six peptides were separated by HPLC and sequenced by Edman degradation, providing sequence data for 83 residues. Data-base search showed similarity of pNiXc to eukaryotic aldolases, with 96% identity to human aldolase A. pNiXc demonstrated aldolase activity with fructose 1,6-bisphosphate as substrate (Km, 30 microM Vmax 26 mumol min-1 mg-1); the aldolase activity was inhibited non-competitively by Cu2+, Cd2+, Co2+, or Ni2+. Equilibrium dialysis showed high affinity binding (Kd, 7 microM) of 1 mole of Ni per mole of 40 kDa subunit. Based on metal-blot competition assays, the abilities of metals to compete with 63Ni2+ for binding to pNiXc were ranked: Cu2+ >> Zn2+ > Cd2+ > Co2+. This study identifies pNiXc as the monomer of fructose-1,6-bisphosphate aldolase A, and raises the possibility that aldolase A is a target enzyme for metal toxicity.
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PMID:The 40 kDa 63Ni(2+)-binding protein (pNiXc) on western blots of Xenopus laevis oocytes and embryos is the monomer of fructose-1,6-bisphosphate aldolase A. 787 95

2-Ethynylnaphthalene (2EN) is a mechanism-based inactivator of rat cytochrome P450 (P450) 2B1 with 1.3 mol of adduct bound per mole of P450 inactivated [Roberts, E.S., Hopkins, N.E., Alworth, W.L., & Hollenberg, P.F. (1993) Chem. Res. Toxicol. 6, 470-479]. Further studies have shown that 2EN is also an efficient mechanism-based inactivator of the 7-ethoxycoumarin O-deethylase activity of rabbit P450 2B4 with 0.83 mol of adduct bound per mole of P450. Cleavage of [3H]2EN-inactivated 2B1 with cyanogen bromide, separation of the peptides by HPLC, and further purification of the radiolabeled fraction by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) led to the identification by autoradiography of a radiolabeled peptide (M(r) approximately 3000). Amino acid sequence analysis of the first 12 N-terminal residues revealed the sequence ISLLSLFFAGTE corresponding to positions 290-301 in the protein. When the radiolabeled fraction from the HPLC separation was analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), peaks at m/z 2722.5 and 2890.6 were detected. The lower mass peak corresponds to the molecular ion (average mass) of the cyanogen bromide peptide Ile290 to Met314 (theoretical 2722.2), while the higher mass peak corresponds to the same peptide with a bound 2-naphthylacetyl group (theoretical 2890.4). When [3H]2EN-inactivated 2B4 was treated with cyanogen bromide, the peptides were separated by HPLC, and the fractions were analyzed by Tricine-SDS-PAGE, two radiolabeled peptides (M(r) = 5000 and 8000) were identified by autoradiography. Amino acid sequence analysis of the first 11 residues revealed identical N-termini with the sequence EKDKSDPSSEF corresponding to positions 273-283.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of active-site peptides from 3H-labeled 2-ethynylnaphthalene-inactivated P450 2B1 and 2B4 using amino acid sequencing and mass spectrometry. 814 77


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