Characterization of phytol-phytanate conversion activity in rat liver

Abstract

The enzymatic conversion of phytol to phytanic acid was investigated in rat liver postnuclear and other subcellularp fractions using [1- 3H]phytol as the substrate. The assay method involved incubation of the substrate with appropriate cofactors and the enzyme source, followed by subjecting the mixture to Folch partition and measuring the radioactivity in the upper layer. The phytol-phytanate conversion activity was present in mitochondrial and microsomal fractions. Cytosol had no activity. In mitochondrial fraction, investigation of cofactor requirements indicated that only NAD was required for activity. Other pyridine nucleotides supported the activity to a lesser extent when compared with NAD. FAD at 1 mM concentration did not support the activity. Bovine serum albumin (0.4 mg/ml) stimulated the activity. The reaction did not require molecular oxygen. From substrate kinetic studies, an apparent K m of 14.3 and 11.1 μM was calculated for phytol in mitochondrial and microsomal fractions, respectively. The amount of tritiated water produced from incubation increased linearly up to 7–8 min. The activity was the amoun of mitochondrial and microsomal protein up to 200 and 40 μg, respectively. Among the various rat tissue homogenates tested, liver had the highest activity. Spleen and kidney had 8–9% of the activity of liver. Brain possessed negligible activity. Both ethanol and pyrazole had no inhibitory effect on phytol-phytanate conversion. This observation and the absence of activity in cytosol suggests that alcohol dehydrogenase may not be involved in phytol-phytanate conversion.