Extraction and detergent/lipid activation of dolichol kinase.
The CTP-dependent dolichol kinase from bovine liver microsomes was optimally extracted using either 0.5% sodium deoxycholate or 0.5% Triton X-100 containing 0.5 M NH4Cl. All activity was found in the supernatant fraction following high-speed centrifugation. This fraction was depleted of phospholipid (phospholipid remaining, less than 5% of total) by gel chromatography of the 0.5% deoxycholate extract. This partially purified enzyme was maximally activated 9- or 53-fold over controls in the presence of 0.1% deoxycholate or 0.1% Triton X-100, respectively. Stimulation of the kinase was also observed with mixtures of dimyristoylphosphatidylcholine and deoxycholate. The level of stimulation by these mixtures was up to 20-fold higher than that observed in controls having deoxycholate alone. Dimyristoylphosphatidylcholine alone was not stimulatory. A 1:1 molar ratio of Triton X-100 or deoxycholate to dimyristoylphosphatidylcholine was optimal for enzyme activation. The half-maximum velocity of the dephospholipidated enzyme at 1:1 molar ratio of detergent to dimyristoylphosphatidylcholine was obtained at 150 or 550 microM CTP in the presence of deoxycholate or Triton X-100, respectively. It has been observed, therefore, that dolichol kinase may be extracted from liver microsomes, depleted of endogenous phospholipids and activated by specific molar ratios of detergent to phospholipid.
D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D003570:Cytidine Triphosphate / Q000494:pharmacology; D003840:Deoxycholic Acid / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D017830:Octoxynol; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011092:Polyethylene Glycols / Q000494:pharmacology
D002413:Cations, Divalent; D003902:Detergents; D003840:Deoxycholic Acid; D011092:Polyethylene Glycols; D003570:Cytidine Triphosphate; D017830:Octoxynol; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020087:dolichol kinase