A B S T R A C T An antibody
METHODSSupernatant fractions were prepared from homogenates of liver and 16 other tissues from 32 adult Sprague-Dawley, 2 Long-Evans, and 2 Gunn (glucuronyltransferase-deficient) (12) rats of either sex. Supernatant fractions were also obtained from at least two specimens of liver from Hartley guinea pigs, albino mice, Rhesus monkeys, Shetland ponies, chickens, rabbits, four species of fish, five species of amphibia, and four species of reptiles. Histologically normal human liver was obtained within 4 hr after patients died from acute myocardial infarction. All laboratory animals were anesthetized with ether and killed immediately; fishes, reptiles, and amphibia were anesthetized with urethane; and horses were killed by captive-bolt after curare administration. Various tissues were rapidly removed, perfused, where possible, with ice-cold 0.9%o saline, and a 25%o homogenate was prepared in 0.25 M sucrose-0.01 M phosphate buffer, pH 7.4, using a motor-driven, teflon pestle, glass homogenizer (Arthur H. Thomas Co., Philadelphia, Pa.). Kidney and muscle required initial disruption in a VirTis (The VirTis Co., Gardiner, N. Y.) homogenizer. Small intestine was removed from duodenum to terminal ileum, washed with cold 0.9% saline, opened lengthwise, and the mucosa was scraped between two glass slides. Blood was collected and permitted to clot at room temperature; sera were stored at -200C. Bile, obtained from the cannulated common bile duct of lightly ether-anesthetized Sprague-Dawley rats, was centrifuged at 2500 g for 30 min. The clear supernate was passed through a Sephadex G-25 column, equilibrated with SPB, to remove bile acids (13). The protein eluted in the void volume was concentrated by ultrafiltration to 1.5 mg protein/ml.