Properties of brain dolichol kinase activity solubilized with a zwitterionic detergent.
Dolichol kinase activity is effectively solubilized by extracting calf brain microsomes with 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent. The solubilized kinase catalyzes the enzymatic phosphorylation of dolichols with either CTP or dCTP serving as phosphoryl donor in the presence of Ca2+. Similar Km values were calculated for CTP (7.7 microM) and dCTP (9.1 microM). Dolichol phosphorylation was inhibited by CDP and dCDP, but not CMP, ADP, GDP, or UDP. A kinetic analysis of the inhibitory effect of CDP revealed a pattern characteristic of competitive inhibition. Dolichol kinase activity was markedly stimulated by the addition of R-dolichol (C95) or S-dolichol(C95). The apparent Km value for R-dolichol(C95) and S-dolichol(C95) was 9 microM, but the Vmax for the phosphorylation reaction was 40% higher with S-dolichol(C95). Incubation of the CHAPS extract with [gamma-32P]CTP and exogenous undecaprenol(C55) resulted in the enzymatic synthesis of a radiolabeled product that was mild acid-labile and chromatographically identical to undecaprenyl monophosphate. An enzymatic comparison with a variety of polyprenol substrates indicates that the solubilized kinase prefers long-chain (C90-95) polyprenols with saturated alpha-isoprene units. The effect of exogenous phosphoglycerides on the kinase activity in the dialyzed CHAPS extracts has also been evaluated. These studies describe the properties and polyprenol specificity of stable, solubilized preparations of dolichol kinase that should be useful for further purification of the enzyme.
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D000818:Animals; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D002413:Cations, Divalent / Q000502:physiology; D002417:Cattle; D002793:Cholic Acids; D003902:Detergents; D008861:Microsomes / Q000201:enzymology; D009711:Nucleotides / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000494:pharmacology; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D012995:Solubility; D013379:Substrate Specificity
D002413:Cations, Divalent; D002793:Cholic Acids; D003902:Detergents; D009711:Nucleotides; D010712:Phosphatidic Acids; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020087:dolichol kinase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate