Determination of peroxisomal fatty XXXX oxidase activity using a @entity35110 -based fluorometric assay.

@entity35110 @entity274 @entity8595 @entity33902 @entity11668
A simple, sensitive fluorometric method for the determination of peroxisomal fatty @entity8595 oxidase (EC 1.3.99.3) activity has been developed. Studies of enzyme activity relative to subcellular distribution and to clofibrate induction indicate that this assay is specific for peroxisomal fatty @entity8595 oxidase. The @entity35110 -dependent production of @entity274 is quantitated by measuring the oxidation of @entity33902 to a fluorescent product in a horseradish peroxidase-coupled assay. Assays can be performed in either a fixed time or continuous mode. In either mode, @entity274 production is related to a change in fluorescence intensity through use of a standard curve generated with known amounts of @entity274 . The use of @entity35110 (12:0), rather than the more generally used substrate @entity11668 (16:0), provides significant advantages. Much of the substrate inhibition problem associated with @entity11668 has been avoided, and a greater than 4.5-fold higher specific activity has been achieved compared with a @entity11668 -based assay. In the fixed-time mode, linearity relative to time and to the amount of enzyme added has been established without resorting to the use of bovine serum albumin as a substrate binding medium. Sensitivity is estimated to be at least equal to that of the most sensitive methods reported, while reliability, versatility and range have been improved. Use of this method should greatly facilitate the study of peroxisomal beta-oxidation regulatory mechanisms in hepatocyte cell culture systems as well as in other circumstances where low activities or small samples must be assayed.
