The Distinctive Occurrence of Acid-Soluble Inosine in Rat Liver Microsomal Preparations

I t has recently been shown that freshly prepared rat liver mitochondria contain a distinct and constant complement of various acid-soluble nucleotides (1). Similar extracts of rat liver microsomes exhibit an entirely different picture, containing in measurable amounts only the nudeoside, inosine. In some recent studies young, Sprague-Dawley rats, obtained from the Holtzman Rat Co., Madison, Wisconsin, were decapitated, the livers excised, and approximately 12 gin. of wet weight immediately homogenized in cold 0.25 ~r sucrose solution. The nuclei, mitochondria, and debris were removed by centrifugation at 9000 g for 15 minutes in the Spinco centrifuge kept at 0°C. The supernatant was carefully removed, leaving behind the loosely packed layer lying on top of the mitochondria. The centrifugation of the supernatant at 105,000 g for 60 minutes gave a translucent reddish pellet which was then taken up in cold 0.25 ~ sucrose solution. Cold perchloric acid was then added to a final concentration of 0.5 M, and the precipitated protein suspension was left in the cold room for a t least 2 hours. The protein was then sedimented, and to the superuatant solution was added cold 1.5 ~r KOH solution to the neutral point of phenol red. The resulting suspension of KC104 was left in the cold room overnight, then centrifuged, and the supernatant solution made slightly alkaline with a drop of NI-I4OH. This solution was finally passed through a dowex