A novel purification and some properties of rat liver mitochondrial choline dehydrogenase.
Choline dehydrogenase (choline:(acceptor) oxidoreductase, EC 1.1.99.1) was purified from rat liver mitochondria. An approx. 240-fold purification was achieved by chemically modified enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) through columns of DEAE-Sepharose CL-6B and the choline-Sepharose 4B with C3-spacer, and after the subsequent release of the thionitrobenzoate with dithiothreitol, through a second column of DEAE-Sepharose CL-6B in the presence of 0.1% Triton X-100. The purified preparation gave a specific activity of 6.3 mumol of O2 consumed/min per mg protein at 30 degrees C with phenazine methosulfate as the primary electron acceptor. After polyacrylamide gel electrophoresis in the presence of 0.5% sodium dodecyl sulfate, the enzyme showed activity in the gel. The preparation thus purified oxidized only choline and betaine aldehyde. The Km value for choline was 7.0 mM at an infinite concentration of phenazine methosulfate at pH 7.6 and 30 degrees C. 2-Dimethyl aminoethanol (Ki,app = 1.0 mM) and monoethanolamine did not work as substrates, inhibiting the enzyme competitively. The absolute requirement of any electron acceptor other than the molecular oxygen was confirmed. The Km value for phenazine methosulfate was about 1.1 mM at infinite concentration of choline. These findings suggested that coenzyme Q served as the primary electron acceptor in vivo.
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001622:Betaine / Q000031:analogs & derivatives / Q000378:metabolism; D002794:Choline / Q000378:metabolism; D050545:Choline Dehydrogenase; D002847:Chromatography, Agarose; D004228:Dithionitrobenzoic Acid; D008773:Methylphenazonium Methosulfate / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D051381:Rats; D013379:Substrate Specificity
D008773:Methylphenazonium Methosulfate; D001622:Betaine; C026820:betaine aldehyde; D004228:Dithionitrobenzoic Acid; D000429:Alcohol Oxidoreductases; D050545:Choline Dehydrogenase; D002794:Choline