Characterization of dolichol kinase from bovine thyroid microsomes.
Bovine thyroid microsomes are able to phosphorylate exogenous [1-3H]dolichol as well as endogenous dolichol. 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 [gamma-32P]CTP. The dolichol kinase activity was not linear with respect to time and exhibited a neutral pH-optimum. Product formation was directly proportional to microsomal protein concentration up to 2.5 mg protein/incubation. The enzyme was found to depend on divalent cations for activity: Mg2+-ions being much more effective than Ca2+- and Mn2+-ions. In accordance, EDTA was strongly inhibitory. The enzyme exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. The apparent Km-value for exogenous dolichol amounted to 4 microM. Those for CTP were estimated to be 3.88 and 10.75 mM with exogenous [1-3H]dolichol depending on the source of CTP. With endogenous dolichol Km-values for CTP of 27.8 and 6.1 microM were calculated in respectively the absence and presence of 5 mM VO4(3-). 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 [gamma-32P]CTP dolichol kinase detergent was only of minor influence (30% stimulation at 0.02% Triton X-100). The levels of the enzymatic activity could be doubled by the inclusion of 18-21 mM NaF [( 1-3H]dolichol kinase) as phosphatase inhibitor: VO4(3-) had practically no effect. In contrast with [gamma-32P]CTP dolichol kinase, the enzymatic activity could be enhanced 4-fold by addition of 5 mM VO4(3-) while F- resulted into no appreciable effect.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D004795:Enzyme Stability; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D009994:Osmolar Concentration; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013961:Thyroid Gland / Q000201:enzymology*
D002413:Cations, Divalent; D013439:Sulfhydryl Reagents; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020087:dolichol kinase