Properties of a dolichol phosphokinase activity associated with rat liver microsomes.
Rat liver microsomes show a capacity to synthesize [1-3H]dolichyl phosphate from [1-3H]-dolichol. Formation of [1-3H]dolichyl phosphate increased continuously over 15 min although the reaction rate was never completely linear. Product formation was directly proportional to microsomal protein concentration between 1.1 mg/mL and the highest concentration tested, 5.5 mg/mL. The reaction rate was linear with respect to the dolichol content of the assay mixture to a saturation point (120 microM). An apparent Km of 50 microM was established for dolichol. The normal phosphate donor for the reaction is CTP and not ATP. The optimum concentration of CTP was 10 mM, and an apparent Km of 4 mM was calculated for this nucleoside triphosphate. The reaction was totally dependent on divalent metal ion, magnesium being more effective than calcium. The optimum concentration of magnesium ion and CTP were the same (10 mM), suggesting that MgCTP2- is utilized as the normal enzyme substrate. Activity measured in the absence of Triton X-100 was only 5% of the activity observed at the optimum (0.5% w/v) detergent concentration. The measurable levels of dolichol phosphokinase could be doubled by the inclusion of 10-15 mM NaF as phosphatase inhibitor. Optimal enzymatic activity was obtained between pH 7.0 and pH 7.5 and could be inhibited by EDTA. The sulfhydryl reagent DTT was slightly stimulatory while the product of the reaction, dolichyl phosphate, was noninhibitory at the highest concentration tested (13.8 microM). The second reaction product (CDP) inhibits the enzymatic phosphorylation of dolichol.
D000818:Animals; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004288:Dolichol Phosphates / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D012265:Ribonucleotides / Q000494:pharmacology
D004288:Dolichol Phosphates; D012265:Ribonucleotides; D003570:Cytidine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020087:dolichol kinase; D008274:Magnesium