Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.2.13 (aldolase)
3,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding isotherms of native bovine serum albumin with cationic detergents, such as octyl, decyl, dodecyl and tetradecylpyridinium bromides were determined at pH 6.8 and 3.4 at 25 degrees C. The isotherms for dodecyl and tetradecylpyridinium bromides were also determined at 3 degrees C. The average number of detergent cations bound increased with increasing hydrocarbon chain length. At low detergent concentration the binding of all alkylpyridinium bromides was smaller at pH 3.4 than at pH 6.8. Dodecylpyridinium bromide was bound to native beta-lactoglobulin, aldolase, ovalbumin, haemoglobin, myoglobin, lysozyme, trypsin and ribonuclease at pH 6.8. No binding occurred to alpha-chymotrypsin and chymotrypsinogen. The free enthalpy change, --delta G degrees, calculated from intrinsic association constants K was determined.
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PMID:Protein-cationic detergent interaction. Equilibrium dialysis study of the interaction of bovine serum albumin and other proteins with alkylpyridinium bromide. 49 43

Nuclear magnetic quadrupole relaxation appears to be a general method for studying the binding of anions to proteins. This is shown by the increase in transverse quadrupole relaxation rate of 35Cl- and 81Br- in the presence of horse liver alcohol dehydrogenase, lysozyme, trypsin, alpha-chymotrypsin, human carbonic anhydrase, fructose-1,6-bisphosphate aldolase and human serum albumin. Of the many possible binding sites at the surface of a protein (e.g. positively charged amino acid side-chains) only a few account for the main part of the relaxation enhancement. This is shown by the decrease in 35Cl- and 81Br- relaxation rate on addition of functional ligands. Large, kinetically inert, complex anions like Pt(CN)2-4 and Au(CN)-2 are found to act as strong competitors towards halogen ions for the high-affinity anion binding sites of a number of proteins. Titrations with complex anions following the 35Cl- or 81Br- relaxation rates are found to be helpful in attempts to elucidate binding mechanisms. Especially, the complex anions may be useful probes for the discrimination between general and metallic anion binding sites in proteins and they also permit correlation of information from X-ray investigations of crystals with that from physical measurements in solution. From the change in halide ion quadrupole relaxation rate on addition of strongly binding ligands the quadrupole coupling constants of the high affinity Cl- and Br- binding sites are estimated using certain assumptions. It is found that for several proteins, comprising the metal-free proteins but also alcohol dehydrogenase and Escherichia coli alkaline phosphatase, the 35Cl quadrupole coupling constants have approximately the same values. For some other metallo-proteins like carbonic anhydrase and a zinc - serum-albumin complex considerably greater quadrupole coupling constants were obtained. The estimated quadrupole coupling constants are used as a basis for a discussion of the interactions involved in anion-protein interactions.
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PMID:Pt(CN)2-4 and Au(CN)-2: potential general probes for anion-binding sites of proteins. 35Cl and 81Br nuclear-magnetic-resonance studies. 120 23

This investigation was undertaken to develop cytotoxicity assay systems using primary cultures of rabbit corneal epithelial cells as an experimental model to evaluate oculotoxic agents and the ability of these in vitro assay systems to predict irritancy potential and delayed toxicity. We have characterized the epithelial nature of the cultures by identifying keratins with antikeratin antibodies (AE1/AE3) and by demonstrating metabolic enzymes important to the integrity of the cells: lactate dehydrogenase, glucose 6-phosphate dehydrogenase and aldolase. Eight surfactants were compared and ranked according to their cytotoxic potential. We evaluated cytotoxicity by measuring leakage of the cytosolic enzyme, lactate dehydrogenase, into the medium, by making morphological observations and by assessing lysosomal neutral red uptake and mitochondrial 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. The cells were treated for 1 h with the surfactants and the possibility of delayed toxicity was evaluated 24 h after removal of the surfactant. The cytotoxicity of the different types of surfactants as shown by all the tests was cationic > anionic = amphoteric > non-ionic. Triton X-100, a non-ionic surfactant but a severe irritant, had a ranking similar to anionic surfactants. The in vitro rankings corresponded well to reported in vivo Draize rabbit eye test data. The 24-h test for lactate dehydrogenase leakage showed that mild and non-irritating surfactants did not demonstrate any subsequent damage after a 1-h exposure, but the extreme and severe surfactants continued to show further damage after the 1-h exposure. These in vitro findings were similar to reported in vivo results. The neutral red and MTT tests did not adequately predict the prolonged toxicity of the more irritating surfactants, as was demonstrated by the lactate dehydrogenase leakage test. We conclude that in vitro cytotoxicity assays using primary cultures of rabbit corneal epithelial cells may be used to rank the cytotoxic potential of surfactants, but only the lactate dehydrogenase leakage test was able to assess prolonged cell injury.
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PMID:Evaluation of surfactant cytotoxicity potential by primary cultures of ocular tissues: I. Characterization of rabbit corneal epithelial cells and initial injury and delayed toxicity studies. 128 45

The complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli has been established in the following manner. After being reduced with dithiothreitol, the purified aldolase was alkylated with iodoacetamide and subsequently digested with trypsin. The resulting 19 peptide peaks observed by high performance liquid chromatography, which compared with 21 expected tryptic cleavage products, were all isolated, purified, and individually sequenced. Overlap peptides were obtained by a combination of sequencing the N-terminal region of the intact aldolase and by cleaving the intact enzyme with cyanogen bromide followed by subdigestion of the three major cyanogen bromide peptides with either Staphylococcus aureus V8 endoproteinase, endoproteinase Lys C, or trypsin after citraconylation of lysine residues. The primary structure of the molecule was determined to be as follows. (formula; see text) 2-Keto-4-hydroxyglutarate aldolase from E. coli consists of 213 amino acids with a subunit and a trimer molecular weight of 22,286 and 66,858, respectively. No microheterogeneity is observed among the three subunits. The peptide containing the active-site arginine residue (Vlahos, C. J., Ghalambor, M. A., and Dekker, E. E. (1985) J. Biol. Chem. 260, 5480-5485) was also isolated and sequenced; this arginine residue occupies position 49. The Schiff base-forming lysine residue (Vlahos, C. J., and Dekker, E. E. (1986) J. Biol. Chem. 261, 11049-11055) is located at position 133. Whereas the active-site lysine peptide of this aldolase shows 65% homology with the same peptide of 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida, these two proteins in toto show 49% homology.
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PMID:The complete amino acid sequence and identification of the active-site arginine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase. 313 64

Steady-state kinetic measurements have shown that 8-azido-1,N6-ethenoadenosine 5'-triphosphate (8-N3-epsilon ATP) can be noncovalently bound to rabbit muscle fructose 1,6-bisphosphate aldolase with Ki = 0.075 mM at pH 8.5. This binding is purely competitive with substrate and occurs at the strong binding site for mononucleotides. Photoaffinity labeling of aldolase in the presence of 8-azido-1,N6-ethenoadenosine 5'-triphosphate results in inactivation of the enzyme. Aldolase is protected against modification in the presence of the inhibitors hexitol 1,6-bisphosphate or ATP. The labeling is saturable, and a good correlation is observed between the loss of enzymatic activity and the incorporation of 8-N3-epsilon ATP into aldolase. In addition, aldolase loses its ability to bind to phosphocellulose following modification. Digestion of labeled protein with trypsin, chymotrypsin, and cyanogen bromide revealed substantial modification of peptide 259-269. Thr-265 was identified as the residue that was covalently modified by 8-N3-epsilon ATP. On the basis of these results and other data we propose a model for the mononucleotide binding site.
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PMID:Photoaffinity labeling of rabbit muscle fructose-1,6-bisphosphate aldolase with 8-azido-1,N6-ethenoadenosine 5'-triphosphate. 365 92

A rat brain S100-binding protein, R40,000, has been isolated, characterized, and identified as fructose-1,6-bisphosphate aldolase. R40,000 was purified by ammonium sulfate precipitation, hydroxylapatite chromatography, dye-binding chromatography, and electroelution from sodium dodecyl sulfate-polyacrylamide gels. Microsequence analysis of a fragment of R40,000 revealed a 15-residue amino acid sequence which shows a high degree of homology to the amino acid sequence of fructose-1,6-bisphosphate aldolase from rabbit muscle and rat liver. Further characterization demonstrated that R40,000 has an amino acid composition, subunit molecular weight, and cyanogen bromide map similar to aldolase. In addition, purified aldolase interacts with S100 alpha and S100 beta by gel overlay, and aldolase enzyme activity is stimulated 2-fold in vitro by S100 alpha and S100 beta. S100 interacts predominantly with the C or brain-specific form of the enzyme in gels and stimulates the activity of the C-enriched form of the enzyme in a calcium-dependent manner. Altogether, these data suggest that fructose-1,6-bisphosphate aldolase may be an intracellular target of S100 action in brain.
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PMID:Identification of a molecular target for the calcium-modulated protein S100. Fructose-1,6-bisphosphate aldolase. 373 59

1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-(14)C]acetic acid in 8m-urea. These peptides have been characterized by amino acid and end-group analysis and their location within the cyanogen bromide cleavage fragments of the enzyme has been determined. 2. Reaction of native aldolase with 5,5'-dithiobis-(2-nitrobenzoic acid), iodoacetamide and N-ethylmaleimide showed that a total of three cysteine residues per subunit of mol.wt. 40000 were reactive towards these reagents, and that the modification of these residues was accompanied by loss in enzymic activity. Chemical analysis of the modified enzymes demonstrated that the same three thiol groups are involved in the reaction with all these reagents but that the observed reactivity of a given thiol group varies with the reagent used. 3. One reactive thiol group per subunit could be protected when the modification of the enzyme was carried out in the presence of substrate, fructose 1,6-diphosphate, under which conditions enzymic activity was retained. This thiol group has been identified chemically and is possibly at or near the active site. Limiting the exposure of the native enzyme to iodoacetamide also served to restrict alkylation to two thiol groups and left the enzymic activity unimpaired. The thiol group left unmodified is the same as that protected by substrate during more rigorous alkylation, although it is now more reactive towards 5,5'-dithiobis-(2-nitrobenzoic acid) than in the native enzyme. 4. Conversely, prolonged incubation of the enzyme with fructose 1,6-diphosphate, which was subsequently removed by dialysis, caused an irreversible fall in enzymic activity and in thiol group reactivity measured with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. It is concluded that the aldolase tetramer contains at least 28 cysteine residues. Each subunit appears to be identical with respect to number, location and reactivity of thiol groups.
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PMID:The reactivity of thiol groups and the subunit structure of aldolase. 542 37

Hemoglobin, aldolase and glyceraldehyde 3-phosphate dehydrogenase are known to bind to the cytoplasmic domain of band 3 protein. Binding of glycolytic enzymes to band 3 protein is inhibited by its amino-terminal fragments. To precisely localize the sequence portion of band 3 protein to which hemoglobin binds and to see whether the same region of amino-acid sequence binds both hemoglobin and glycolytic enzymes, a simple, direct solid-phase binding assay was developed. Peptides generated from the 23-kDa fragment by trypsin, cyanogen bromide and mild acid hydrolysis were used as inhibitors to determine the minimal sequence structure involved in the binding of the 23-kDa fragment to hemoglobin. The shortest peptide which inhibits the binding of the 23-kDa fragment is an acid cleavage peptide containing the sequence positions 1 to 23. This sequence is unusual as 14 of its residues are negatively charged, it contains no basic residues and has its amino terminus blocked. Using aldolase, glyceraldehyde-3-phosphate dehydrogenase and hemoglobin as competitive inhibitors in the binding of 23-kDa fragment, the affinity of hemoglobin to this fragment appears several-fold weaker than that of both the enzymes. These findings demonstrate that glycolytic enzymes and hemoglobin bind competitively to the same polyanionic sequence region of band 3 protein.
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PMID:Hemoglobin binds to the amino-terminal 23-residue fragment of human erythrocyte band 3 protein. 671 38

The inactivation of Streptococcus faecalis by radiolytically generated selective inorganic radical anions was investigated. The Br-2 radical, but not (CNS)-2, had a pronounced radiosensitizing action. In gamma-irradiated solutions at pH 7.0, the radiosensitization of a variety of scavenging systems was studied. Among these the D10 for N2/Br- was 0.082 kGy while N2O/CNS- = 0.35 kGy, N2O = 0.25 kGy, N2 = 0.47, and O2 = 0.16 kGy. As shown previously, inactivation in N2O/Br- systems is due mainly to Br2 and HOBr. From the variation of the inactivation with pH by Br-2 and (CNS)-2 it was deduced that tyrosine is crucial for the survival of S. faecalis via inactivation of enzymes with essential tyrosine residues such as aldolase and lipoyl dehydrogenase which are presumably needed to make energy available for DNA repair. Studies with a variety of scavengers also revealed that the t-butanol radical produced some radiosensitization of S. faecalis while the damaging effect of e-aq was much less than OH as shown by the D10 at pH 7.0; N2/t-butanol = 0.32 and N2/ethanol = 0.71. The radiosensitizing action of Br-2 in a natural environment containing sewage sludge was also determined, using the faecal streptococcal group as test organisms.
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PMID:Radiosensitization of microorganisms by radical anions. II. Streptococcus faecalis. 679 2

E. coli pyruvate oxidase (pyruvate:ferricytochrome b1 oxidoreductase, EC 1.2.2.2) is a peripheral membrane flavoenzyme which has been purified to homogeneity. In vivo the oxidase resides on the inner surface of the cytoplasmic membrane and is coupled to the bacterial electron transport chain. In vitro, the purified oxidase requires lipids for full enzymatic activity. Previous studies have characterized the conformational and energetic coupling between the lipid-binding site(s) and the catalytic active site. The affinity of the enzyme for phospholipids and detergents is significantly enhanced when the flavoprotein is in the reduced form, i.e., in the presence of pyruvate and the required cofactor, thiamin pyrophosphate. The lipid-binding studies were hindered due to the complicating factor of the self-association of the substrate-reduced flavoprotein. In this paper, fluorescence techniques are employed to measure the binding of a detergent-like activator to the oxidase. The experiments are performed at much lower protein concentrations than previously employed, so that protein aggregation is not a problem. The chromophore on the activator, 2-(N-decyl)aminonaphthalene-6-sulfonic acid is effective at quenching the pyruvate oxidase intrinsic tryptophan fluorescence. Quenching titrations are used to obtain the binding isotherm. AT DNS concentrations less than 10(-5) M, the results show a larger amount of DNS binding to the reduced flavoprotein than to the oxidized form of the enzyme. This is the concentration range where DNS is an effective activator of the enzyme. This represents a class of binding sites specifically found on pyruvate oxidase and not apparent in other proteins such as lysozyme or aldolase. At the DNS concentration which is optimum for activation approx. 20 molecules of DNS are bound per enzyme tetramer in the absence of the substrate. The pyruvate-reduced form of the enzyme binds about 40--50 molecules of DNS per tetramer. Qualitatively, the results are similar to what was previously found for both sodium dodecyl sulfate and cetyl trimethylammonium bromide. However, in both these cases, the amount of bound detergent was nearly an order of magnitude less than the values obtained using DNS.
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PMID:The binding of a fluorescent activator 2-(N-decyl)aminonaphthalene-6-sulfonic acid to pyruvate oxidase. 700 Jan 89


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