HPLC-UV method for measuring nicotinamide N-methyltransferase activity in biological samples: evidence for substrate inhibition kinetics
| Autor: | Muhammad M. Vasaya, Misha Patel, Richard B. Parsons, Daniel S. Asker |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Niacinamide
Clinical Biochemistry Nicotinamide N-methyltransferase Biochemistry Analytical Chemistry chemistry.chemical_compound Mice Limit of Detection Nicotinamide N-Methyltransferase Animals Humans Aldehyde oxidase Chromatography High Pressure Liquid Enzyme Assays Nicotinamide Substrate (chemistry) Cell Biology General Medicine Molecular biology In vitro Orders of magnitude (mass) Nicotinamide N-methyltransferase activity Kinetics chemistry Liver Specific activity Spectrophotometry Ultraviolet Rabbits |
| Zdroj: | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. |
| ISSN: | 1873-376X |
| Popis: | Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide. Enhanced NNMT activity is a feature of many types of cancer, and has been linked to processes such as tumour metastasis, resistance to radiotherapy and tumour drug resistance. As such, inhibition of NNMT activity is a promising therapeutic target for cancer therapy. To screen for NNMT inhibitors, there is a need for a standardised, rapid and cost-effective NNMT assay. Here, we describe a cell-free assay coupled with ion-pairing reverse-phase HPLC-UV detection of 1-methylnicotinamide which requires minimal sample manipulation, is linear over 2.5 orders of magnitude with limits of detection and quantification of 0.05 and 0.15nmol 1-methylnicotinamide/100μL injection respectively. The assay was sufficiently sensitive to measure basal hepatic 1-methylnicotinamide concentration and NNMT activity in mouse, rabbit and human liver. 1-Methylnicotinamide concentration and the NNMT kinetic parameters specific activity, Vmax and Km all demonstrated species differences. NNMT also demonstrated substrate inhibition kinetics in all three species, which again was species-specific in term of calculated Ki. This assay demonstrates improved sensitivity over other previously published methods whilst lacking many of their drawbacks such as extensive sample preparation, use of non-physiological substrates and radioisotopic labelling. |
| Databáze: | OpenAIRE |
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