Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
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Target Concepts:
Gene/Protein
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Query: EC:1.14.11.2 (
prolyl hydroxylase
)
1,814
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
An assay for
prolyl 4-hydroxylase
(
EC 1.14.11.2
) is described which measures
succinic acid
produced during the decarboxylation of 2-oxoglutaric acid in the presence of poly(L-Pro-Gly-L-Pro). [1-14C]Succinic acid was separated from its precursor 2-oxo[5-14C]glutaric acid by using ion-exchange minicolumns. The contamination of
succinic acid
by 2-oxoglutaric acid was approx. 1%, and the recovery of
succinic acid
was 100%. Kinetic parameters of
prolyl 4-hydroxylase
measured by the assay showed good agreement with published values. Our experience indicates that the measurement of
prolyl 4-hydroxylase
by the production of
succinic acid
is especially suited to investigations involving large numbers of assays.
...
PMID:Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns. 302 79
The prolyl-4-hydroxylase proteins regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylation of proline residues targeting HIF-1alpha for proteasomal degradation. Using the purified catalytic domain of
prolyl hydroxylase
2 (PHD2(181-417)), an enzymatic assay has been developed to test inhibitors of the enzyme in vitro. Because PHD2 hydroxylates HIF-1alpha, with
succinic acid
produced as an end product, radiolabeled [5-(14)C]-2-oxoglutaric acid was used and formation of [14C]-
succinic acid
was measured to quantify PHD2(181-417) enzymatic activity. Comparison of the separation of 2-oxoglutaric acid and
succinic acid
by either ion exchange chromatography or precipitation with phenylhydrazine showed similar results, but the quantification and throughput were vastly increased using the latter method. The PHD2 reaction was substrate and concentration dependent. The addition of iron to the enzyme reaction mix resulted in an increase in enzymatic activity. The Km value for 2-oxoglutaric acid was determined to be 0.9 microM, and known PHD2 inhibitors were used to validate the assay. In addition, the authors demonstrate that this assay can be applied to other 2-oxoglutaric acid-dependent enzymes, including the asparaginyl hydroxylase, factor-inhibiting HIF-1alpha (FIH). A concentration-dependent increase in
succinic acid
production using recombinant FIH enzyme with a synthetic peptide substrate was observed. The authors conclude that a by-product enzyme assay measuring the conversion of 2-oxoglutaric acid to
succinic acid
using the catalytic domain of the human PHD2 provides a convenient method for the biochemical evaluation of inhibitors of the 2-oxoglutaric acid-dependent hydroxylases.
...
PMID:Characterization of a robust enzymatic assay for inhibitors of 2-oxoglutarate-dependent hydroxylases. 1949 81
