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)

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

Serum sialic acid has been assayed enzymatically. The reaction includes neuraminidase hydrolysis of glycoprotein, cleavage of sialic acid to pyruvate by N-acetyl neuraminic acid (NANA)-aldolase, oxidation of pyruvate by pyruvate oxidase which produces hydrogen peroxide, and colorimetry of hydrogen peroxide using the peroxidase-p-chlorophenol-4-amino-antipyrine method. This method showed good correlation between a chemical method (r = 0.984), good recovery (98.8%) and good reproducibility (within-run-precision: 1.0% C.V.; day-to-day precision: 1.9% C.V). Intrinsic serum pyruvate produces an equimolar positive effect. The normal value range is 1.94 +/- 0.29 mmol/l (mean +/- S.D.,n = 24).
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PMID:Enzymatic assay of serum sialic acid. 747 82

Cancer patients are found to have elevated serum levels of lipid-associated sialic acid (LASA). LASA measurement provides a valuable test for the diagnosis of human cancer. The currently used measurement methods are tedious and nonselective. Here we report a method based on immobilized enzyme minicolumns in a flow system for the analysis of this clinically important analyte. Three enzymes comprising two minicolumns are introduced online and the analyte in a precisely injected volume of 20 microL is made to pass through these immobilized enzyme columns. The final product of the reaction is detected amperometrically. Neuraminidase and NANA-aldolase are coimmobilized on controlled pore glass, followed by a column of pyruvate oxidase. The method was applied to the quantitation of LASA in the sera of normal individuals and patients with cancer of the esophagus. We observed no loss of activity of the immobilized enzymes after analyzing about 300 samples. The sample throughput was 30 samples per hour.
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PMID:Quantitation of the cancer marker LASA using immobilized enzymes in a semiautomated system. 1963 14