Purification and properties of choline kinase from rat brain.
A blue-dye column separated rat brain choline kinase (EC 2.7.1.32) into two peaks, very likely corresponding to distinct isozymes. The major-peak enzyme was purified 15,000-fold to homogeneity. The final specific activity was approx. 40 mumol.min-1.mg-1. This is 10-times higher than that reported for the enzymes from lung and kidney. The purified enzyme gave a single 44 kDa protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Analytical gel-filtration showed that the native enzyme had a molecular weight of 90,000 and a Stokes radius of 4.2 nm. The sedimentation coefficient was deduced to be 4.8 S and the molecular weight 87,600 by sucrose-density-gradient centrifugation. Hence, the native enzyme appears to be a dimer. The apparent Km values for ATP and choline were 1.0 mM and 14 microM, respectively. At high choline concentrations, the enzyme showed deviation from Michaelis-Menten kinetics. The enzyme was active in a high pH range and utilized a variety of amino alcohols structurally related to choline, including ethanolamine, N-methylethanolamine and N,N-dimethylethanolamine as substrates. Spermine and spermidine stimulated the enzyme by decreasing the apparent Km for ATP and increasing Vmax. Although less efficiently, monovalent cations such as NH4+, K+, Li+ and Na+ and quaternary amines such as carpronium, chlorocholine and acetylcholine were also stimulatory.
Uchida
T
T
Department of Biochemistry, Gunma University School of Medicine, Maebashi, Japan.
Yamashita
S
S
D000588:Amines / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002797:Choline Kinase / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification*; D051381:Rats; D013379:Substrate Specificity
D000588:Amines; D046911:Macromolecular Substances; D010770:Phosphotransferases; D002797:Choline Kinase; D008274:Magnesium