Partial purification of two forms of Choline kinase and separation of Choline kinase from sphingosine kinase of rat brain

Abstract

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.

Purification and Characterization of Rat Kidney Sphingosine Kinase

“…We found that sphingosine kinase binds tightly to 1,6-diaminohexane covalently linked to Sepharose 4B (EAHSepharose), but could not be eluted from this matrix by substrates containing primary amino groups, such as sphingosine or choline, in a similar manner, as was previously found for purification of choline kinase (44). In contrast, at least 70% of the applied sphingosine kinase activity was eluted from EAHSepharose with 0.15 M NaCl, whereas only 14 -18% of the applied proteins were eluted in this fraction, resulting in 3-5-fold purification (Fig.…”

“…Similarly, another kinase, choline kinase, has been partially purified from rat kidney by Ͼ200,000-fold (44). This study provides the basis for molecular characterization of sphingosine kinase and will aid in elucidation of its role in various physiological processes.…”

Choline kinase

“…versibility: ? <13>[15,27,41])[15,27,41] P ADP + N-methylethanolamine phosphate S N-monoethylethanolamine + ATP <20> (Reversibility: ? <20>[7])[7] P p-chloromercuribenzoate <19> (<5> no effect…”