Identification of natural compounds inhibiting Trypanosoma brucei Glyceraldehyde-3-phosphate dehydrogenase by in-silico screening and a spectrophotometric enzyme inhibition assay

Planta Medica ◽  
2014 ◽  
Vol 80 (16) ◽  
Author(s):  
F Herrmann ◽  
M Lenz ◽  
TJ Schmidt
Keyword(s):  
Trypanosoma Brucei ◽  
Enzyme Inhibition ◽  
In Silico ◽  
Natural Compounds ◽  
Inhibition Assay ◽  
In Silico Screening ◽  
Enzyme Inhibition Assay

Plasma methotrexate as determined by liquid chromatography, enzyme-inhibition assay, and radioimmunoassay after high-dose infusion.

1980 ◽  
Vol 26 (6) ◽  
pp. 734-737 ◽  
Author(s):  
S K Howell ◽  
Y M Wang ◽  
R Hosoya ◽  
W W Sutow
Keyword(s):  
Liquid Chromatography ◽  
Enzyme Inhibition ◽  
Plasma Concentrations ◽  
Cross Reactivity ◽  
High Dose ◽  
Maximum Plasma ◽  
Inhibition Assay ◽  
Dose Infusion ◽  
Enzyme Inhibition Assay ◽  
Related Compounds

Abstract Three techniques for measuring methotrexate show various cross reactivities with methotrexate-related compounds: “high-pressure” liquid chromatography, by principle, is virtually specific for methotrexate; the enzyme-inhibition assay quantitates methotrexate, methotrexate diglutamate, and methotrexate triglutamate equally well, but has a 10% cross reactivity with 4-amino-4-deoxy-N10-methylpteroic acid and 1% with 7-hydroxymethotrexate; radioimmunoassay shows an equal cross reactivity with methotrexate, 4-amino-4-deoxy-N10-methylpteroic acid, methotrexate diglutamate and triglutamate, and a 5 to 10% cross reactivity with 7-hydroxymethotrexate. Radioimmunoassay almost always yielded the highest values for methotrexate, followed by enzyme-inhibition assay then liquid chromatography. The presence of two methotrexate-related compounds, 7-hydroxymethotrexate and 4-amino-4-deoxy-N10-methylpteroic acid, was confirmed in human urine samples and quantitated in patients’ plasma by liquid chromatography, the respective maximum plasma concentrations being 250 and 16 mumol/L. Materials cross reacting with methotrexate in radioimmunoassay of chromatographic fractions from plasma were also noted in fractions corresponding to methotrexate diglutamate and triglutamate peaks, in quantities estimated to be 47 and 30 nmol/L methotrexate equivalents, respectively. 7-Hydroxymethotrexate is eliminated more slowly than methotrexate and its production increases with dosages of methotrexate.

scholarly journals Pneumococcal Haemophilus influenzae Protein D Conjugate Vaccine Induces Antibodies That Inhibit Glycerophosphodiester Phosphodiesterase Activity of Protein D

2008 ◽  
Vol 76 (10) ◽  
pp. 4546-4553 ◽  
Author(s):  
Maija Toropainen ◽  
Anna Raitolehto ◽  
Isabelle Henckaerts ◽  
Dominique Wauters ◽  
Jan Poolman ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Enzymatic Activity ◽  
Enzyme Inhibition ◽  
Acute Otitis Media ◽  
Vaccine Antigen ◽  
Inhibition Assay ◽  
Igg Antibodies ◽  
Enzyme Inhibition Assay ◽  
Glycerophosphodiester Phosphodiesterase ◽  
Inhibition Index

ABSTRACT Haemophilus influenzae outer membrane protein D (PD) is a glycerophosphodiester phosphodiesterase (GlpQ) activity-possessing virulence factor and a promising vaccine antigen, providing 35.3% efficacy against acute otitis media caused by nontypeable H. influenzae (NTHI) when it was used as a carrier protein in a novel pneumococcal PD conjugate (Pnc-PD) vaccine. To study if PD-induced protection against NTHI could be due to antibodies that inhibit or neutralize its enzymatic activity, a GlpQ enzyme inhibition assay was developed, and serum samples collected from Finnish infants before and after Pnc-PD vaccination were analyzed for enzyme inhibition and anti-PD immunoglobulin G (IgG) antibody concentration. Before vaccination at age 2 months, the majority (84%) of infants (n = 69) had no detectable anti-PD IgG antibodies, and all were enzyme inhibition assay negative (inhibition index, <20). At age 13 to 16 months, all infants receiving three or four doses of Pnc-PD had detectable anti-PD IgG antibodies and 36% (8/22 infants) of the infants receiving three doses and 26% (6/23 infants) of the infants receiving four doses of Pnc-PD were inhibition assay positive (inhibition index, ≥20). No significant rise in anti-PD IgG antibodies or enzyme inhibition among control vaccinees (n = 24) receiving three doses of hepatitis B vaccine was detected. A modest correlation (r s , ∼0.66) between anti-PD IgG concentration and enzyme inhibition was detected; however, their kinetics were clearly different. These data suggest that measurement of antibody responses that inhibit PD's enzymatic activity could be a useful tool for assessing Pnc-PD vaccine-induced protective immunity against NTHI.

Enzyme immunoassay and enzyme inhibition assay of methotrexate, with use of the centrifugal analyzer.

1981 ◽  
Vol 27 (3) ◽  
pp. 380-384 ◽  
Author(s):  
M A Pesce ◽  
S H Bodourian
Keyword(s):  
Enzyme Immunoassay ◽  
Enzyme Inhibition ◽  
Correlation Coefficients ◽  
High Dose ◽  
Inhibition Assay ◽  
High Dose Therapy ◽  
Standard Curve ◽  
Competitive Protein Binding ◽  
Enzyme Inhibition Assay ◽  
Homogeneous Enzyme Immunoassay

Abstract Methotrexate was determined by the homogeneous enzyme immunoassay (EMIT) with the Multistat and CentrifiChem centrifugal analyzers and by the enzyme inhibition assay with use of the Multistat centrifugal analyzer. With both methods, the standard curve extends from 0.2 to 2.0 mumol/L and there is no interference from bilirubin in concentrations up to 100 mg/L. Moderately lipemic samples do not interfere with the EMIT method, but lower the values obtained with the enzyme inhibition assay. Hemoglobin concentrations as great as 1 g/L do not affect results for methotrexate obtained by the enzyme inhibition assay. With the EMIT assay, methotrexate values are lowered in samples containing hemoglobin in concentrations exceeding 750 mg/L. With the EMIT assay, the following compounds in concentrations of 1 mmol/L do not interfere: leucovorin, 5-methyl-tetrahydrofolate, 5-fluorouracil, and 6-mercaptopurine. When folic acid (100 mumol/L) was added to a serum that did not contain methotrexate, a response equivalent to 0.15 mumol/L was obtained. Methotrexate is stable for five days in serum stored at 23, 4, or -20 degrees C. Within-run precision (CV) for the enzyme inhibition method ranged from 4.7 to 8.1% and for the EMIT assay from 2.5 to 6.2%. Methotrexate concentrations in the serum of children receiving high-dose therapy were compared by three methods: competitive protein binding, EMIT, and enzyme inhibition assays. The correlation coefficients averaged 0.95.

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