The distribution of choline oxidase activity in rat liver.

Rat liver was fractionated essentially as described by Hogeboom, Schneider, and Pallade (2). The liver was homogenized in 0.88 M sucrose (1 gm. of liver to 10 ml.) in the cold and centrifuged in 25 ml. Lustroid tubes in a 32” angle centrifuge (Custom Scientific Instruments, Inc.) in a cold room kept at 5”. The homogenate was separated into nuclear, mitochondrial, and supernatant fractions. The nuclear fraction was prepared by centrifugation at 1200 X g for 10 minutes, the mitochondrial fraction by centrifugation at 20,000 X g for 25 minutes. The nuclear fraction was spun down and resuspended three times, the mitochondrial fraction one or two times. The residual supernatant fraction was not further fractionated, as it was found to contain very little of the total activity. Choline oxidase activity was measured manometrically (3), on aliquots containing, or equivalent to, 93 mg. of wet tissue. The reaction mixture consisted of 0.033 M phosphat,e (K) buffer, pH 7.4, 0.00027 M calcium chloride, 0.05 M choline chloride, and 0.000016 M cytochrome c. The cytochrome c was added, as Mann, Woodward, and Quastel (4) showed that choline dehydrogenase is a cytochrome-linked dehydrogenase. Succinoxidase activity was measured as described by Schneider and Potter (5), except that aluminum chloride was omitted.