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Literature DB >> 3028379

Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns.

C J Cunliffe, T J Franklin, R M Gaskell.

Abstract

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.

Entities: Chemical

Mesh：

Substances：

Year: 1986 PMID： 3028379 PMCID： PMC1147460 DOI： 10.1042/bj2400617

Source DB: PubMed Journal: Biochem J ISSN： 0264-6021 Impact factor: 3.857

11 in total

Review 4. Spectroscopic and in vitro Investigations of Fe²⁺ /α-Ketoglutarate-Dependent Enzymes Involved in Nucleic Acid Repair and Modification.

Authors: David Schmidl; Niko S W Lindlar Né Jonasson; Annika Menke; Sabine Schneider; Lena J Daumann
Journal: Chembiochem Date: 2022-02-15 Impact factor: 3.461

4 in total

Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns.

1. ENZYMATIC HYDROXYLATION OF PROLINE IN MICROSOMAL POLYPEPTIDE LEADING TO FORMATION OF COLLAGEN.

2. Modification of the tritium-release assays for prolyl and lysyl hydroxylases using Dowex-50 columns.

3. Enzymatic hydroxylation of proline and lysine in protocollagen.

4. Mechanism of the prolyl hydroxylase reaction. 2. Kinetic analysis of the reaction sequence.

5. Mechanism of the prolyl hydroxylase reaction. 1. Role of co-substrates.

6. Decarboxylation of alpha-ketoglutarate coupled to collagen proline hydroxylase.

7. Properties of prolyl 4-hydroxylase containing firmly-bound iron.

8. An improved method for the purification of vertebrate prolyl hydroxylase by affinity chromatography.

9. Inhibition of prolyl 4-hydroxylase by hydroxyanthraquinones.

10. Hydroxylation of proline in synthetic polypeptides with purified protocollagen hydroxylase.

1. Inhibition of prolyl 4-hydroxylase in vitro and in vivo by members of a novel series of phenanthrolinones.

2. Inhibition of collagen hydroxylation by 2,7,8-trihydroxyanthraquinone in embryonic-chick tendon cells.

3. Direct and continuous assay for prolyl 4-hydroxylase.

Review 4. Spectroscopic and in vitro Investigations of Fe²⁺ /α-Ketoglutarate-Dependent Enzymes Involved in Nucleic Acid Repair and Modification.

Review 4. Spectroscopic and in vitro Investigations of Fe2+ /α-Ketoglutarate-Dependent Enzymes Involved in Nucleic Acid Repair and Modification.

Review 4. Spectroscopic and in vitro Investigations of Fe²⁺ /α-Ketoglutarate-Dependent Enzymes Involved in Nucleic Acid Repair and Modification.