The elution of pristanoyl‐CoA oxidase from butyl‐Sepharose required unusually high concentrations of ethylene glycol, enabling the large‐scale purification of this oxidase in a single chromatographic step. The enzyme, the native molecular mass of which was estimated previously at 415 kDa by gel filtration (Van Veldhoven, P. P., Vanhove, G., Vanhoutte, F., Dacremont, G., Eyssen, H. J. & Mannaerts, G. P. (1991) J. Biol. Chem. 266, 24676–24683), migrated as a 513‐kDa protein during native gel electrophoresis. It showed a typical flavoprotein spectrum and probably binds 4 mol FAD/mol enzyme. Its amino acid composition was different from those of other known acyl‐CoA oxidases. Screening of different rat tissues, either for enzyme activity or by immunoblotting, revealed the highest level of pristanoyl‐CoA oxidase in liver, followed by kidney, intestinal mucosa, spleen and lung. The oxidase activities, measured with 2‐methylpalmitoyl‐CoA as the substrate, in livers from other vertebrates including man were low compared to rat. This was also confirmed by immunoblotting which provided a clear signal only in rat liver, possibly indicating that pristanoyl‐CoA oxidase might be a rat‐specific oxidase.
The acyl-CoA oxidase, catalysing the peroxisomal desaturation of the CoA-ester of trihydroxycoprostanic acid, a bile acid intermediate, has been purified to homogeneity from rat liver. Its native molecular mass, as determined by gel filtration and native gel electrophoresis, was 120 and 175 kDa respectively, suggesting a homodimeric protein consisting of 68.6 kDa subunits. If isolated in the presence of FAD, the enzyme showed a typical flavoprotein spectrum and contained most likely 2 mol of FAD per mol of enzyme. The cofactor, however, was loosely bound. The enzyme acted exclusively on 2-methyl-branched compounds, including pristanoyl-CoA and 2-methylhexanoyl-CoA if albumin was present. Important parameters to obtain a pure and active enzyme were the following: (1) using chromatographic separations like hydrophobic interaction and metal affinity, which allow the presence of high salt concentrations, conditions which stabilize the oxidase; (2) avoiding dialysis and (NH4)2SO4 precipitation; (3) including, when appropriate, FAD, dithiothreitol and a diol-compound in the solvents; and (4) carefully monitoring the removal of other acyl-CoA oxidases which possess the same native molecular mass and subunit size.
The complexity and heterogeneity of cancers leads to variable responses of patients to treatments and interventions. Developing models that accurately predict patient’s care pathways using prognostic and predictive biomarkers is increasingly important in both clinical practice and scientific research. The main objective of the ATHENA project is to: (1) accelerate data driven precision medicine for two use cases – bladder cancer and multiple myeloma, (2) apply distributed and privacy-preserving analytical methods/ algorithms to stratify patients (decision support), (3) help healthcare professionals deliver earlier and better targeted treatments, and (4) explore care pathway automations and improve outcomes for each patient. Challenges associated with data sharing and integration will be addressed and an appropriate federated data ecosystem will be created, enabling an interoperable foundation for data exchange, analysis and interpretation. By combining multidisciplinary expertise and tackling knowledge gaps in ATHENA, we propose a novel federated privacy preserving platform for oncology research.
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