Partial purification of two forms of choline kinase and separation of choline kinase from sphingosine kinase of rat brain.
Choline kinase of rat brain was purified approximately 200,000 fold using acid precipitation, ammonium sulphate fractionation, Q-Sepharose, Octyl-Sepharose and AH-Sepharose chromatography. The ability of this enzyme to catalyze the phosphorylation of choline, ethanolamine (Etn), monomethylethanolamine (MeEtn), dimethylethanolamine (Me2Etn) and sphingosine was investigated. Choline kinase was separated from sphingosine kinase. The fraction with highly purified choline kinase had four major polypeptides with different molecular masses and possessed activities towards choline, Etn, MeEtn and Me2Etn. Two forms of choline kinase were obtained when the enzymatically active fractions eluted from the Q-Sepharose column were subjected to a horizontal isoelectrofocusing electrophoresis. One form focused around pH 4.7 and is able to phosphorylate choline, Etn, MeEtn and Me2Etn. The other form focused around pH 10 and possessed only choline kinase activity. The latter form of choline kinase did not display classical Michaelis-Menten's mechanism but revealed a positive co-operative pattern for two choline binding sites. This form was purified to apparent homogeneity with a approximate molecular mass of 14.4 kDa.
Cao
Z M
ZM
Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Canada.
Kanfer
J N
JN
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002797:Choline Kinase / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D000099157:Sphingosine Kinase
D002797:Choline Kinase; D007527:Isoenzymes; D017853:Phosphotransferases (Alcohol Group Acceptor); D000099157:Sphingosine Kinase