Drug enzyme assays

Enzyme Assay. Na , K -ATPase, and sarcoplasmic reticulum Ca - ATPase were prepared from rat hearts (22) and dog hearts (23), respectively. Bovine heart cyclic AMP phosphodiesterase was purchased from Sigma. The enzyme reaction was carried out after 5-min pretreatment with the drug, and the amount of inorganic phosphate liberated during the reaction period was determined. 

Trubetskoy, O.V., Gibson, J.R., and Marks, B.D. 2005. Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes. J. Biomol. Screen. 10 56. 

Many drugs are effective as a result of inhibiting one or more enzymes. Enzyme inhibitors reduce the rate of formation of product. In a closed system such as an enzyme assay in a test tube, they do not alter the amount of product that is ultimately generated rather, it takes longer in the presence of an inhibitor to generate a given amount of product. 

Fluoroimmunoassays. This assay requires the drug being assayed to be labelled with umbelliferyl-3 D-galactoside. The enzyme 3-galactosidase is added and the fluorescent products are released from the labelled antibiotic. The antibody in the bound fraction... 

Clinical use Naproxen (Todd and Clissold, 1990) is a nonsteroidal anti-inflammatory drug used for the treatment of mild to moderate pain and inflammatory pain conditions such as rheumatoid arthritis, osteoarthritis, soft tissue disorders, postoperative pain and dysmenorrhoea. It is also used to treat migraine. Naproxen shows balanced inhibition of both COX isoenzymes in a cellular assay and a preference for COX-1 in a whole blood assay and in an enzyme assay using recombinant human enzymes. 

Cell Culture. KB cells were maintained in a humidified atmosphere of 5% carbon dioxide - 95% air at 37°C in the presence of modified Eagle s medium containing calf serum (10%), penicillin (100 jug/ml) and streptomycin (100 units/ml). Cells were routinely subcultured with 0.25% trypsin and stocks were discarded after twenty passages. All drugs were administered with fresh media 24 hours after subculture in the following concentrations TPA, 1.6 uM RA, 1.6 juM butyric acid, 2mM. Drug treatments were for 20-24 hours. Cells were harvested for enzyme assays with phosphate-buffered saline containing 0.05% EDTA and stored at -20°C in 0.32 M sucrose. 

The DTE module (Fig. 17a) is an accessory fitting for the assay of electrolytes and can be operated in addition to the main instrument. The structure of the slides differs from that of the slides used for the assay of substances, such as drugs, enzymes or metabolites. These slides are also inscribed on each side with a code that is readable by machine and user. These slides enable the assay of electrolytes by means of single use ion-selective electrodes. 

In addition to commercially production, a great deal of research and development work on biochips has been going on both in industry and in academia. Genomic analysis of DNA and RNA continues to be the focus of interest, but more and more effort is being spent on proteomic analysis of proteins and peptides. Several enzyme assays and immunoassays designed based on microarray-based systems with simple microfluidic control are close to commercialization. They can be a vital tool in clinic diagnostics, drug discovery, and biomedical research. 

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