Characterization of dolichol kinase from bovine thyroid microsomes

Abstract

1. 1. Bovine thyroid microsomes are able to phosphorylate exogenous [1- 3H]dolichol as well as endogenous dolichol. 2. 2. The properties and specificity of the dolichol kinase activity have been studied by following the phosphorylation of [1- 3H]dolichol to [1- 3H]DMP as well as the formation of [ 32P]DMP from endogenous dolichol and [γ- 32P]CTP. 3. 3. The dolichol kinase activity was not linear with respect to time and exhibited a neutral pH-optimum. 4. 4. Product formation was directly proportional to microsomal protein concentration up to 2.5 mg protein/incubation. 5. 5. The enzyme was found to depend on divalent cations for activity: Mg 2+-ions being much more effective than Ca 2+- and Mn 2+-ions. 6. 6. In accordance, EDTA was strongly inhibitory. 7. 7. The enzyme exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. 8. 8. The apparent K m -value for exogenous dolichol amounted to 4 μM. Those for CTP were estimated to be 3.88 and 10.75 mM with exogenous [1- 3H]dolichol depending on the source of CTP. 9. 9. With endogenous dolichol K m -values for CTP of 27.8 and 6.1 μM were calculated in respectively the absence and presence of 5 mM VO 4 3−. 10. 10. Triton X-100 (0.15%) was necessary in the [1- 3H]dolichol kinase assay (only 3% of enzymatic activity in the absence of detergent), while with [γ- 32P]CTP dolichol kinase detergent was only of minor influence (30% stimulation at 0.02% Triton X-100). 11. 11. The levels of the enzymatic activity could be doubled by the inclusion of 18–21 mM NaF ([1- 3H]dolichol kinase) as phosphatase inhibitor; VO 4 3− had practically no effect. 12. 12. In contrast with [γ- 32P]CTP dolichol kinase, the enzymatic activity could be enhanced 4-fold by addition of 5 mM VO 4 3− while F − resulted into no appreciable effect. 13. 13. In both approaches sulphydryl groups were essential for enzymatic activity. 14. 14. Bacitracin inhibited dolichol kinase. 15. 15. Propylthiouracyl, cAMP, TSH and several other agonists were without effect on the system in vitro. 16. 16. VOS, PO 4 3−, P 2O 7 4− and several phosphomonoesters had no influence on the enzymatic activity. [1- 3H]dolichol kinase activity could readily be solubilized (∼70%).