A Stabilizing Factor for Cytoplasmic Nucleoproteins

The macromolecular nudeoproteins of rat liver cytoplasm give rise to a number of ultracentrifugal boundaries with sedi-mentation rates between 107 and 27 S (1). These are probably the same structures which appear as spherical particles 100 to 150 A in diameter in electron micrographs of thin sections of whole tissue (2). They contain about 45 per cent PNA (3). Since the uptake of labelled amino acids is most rapid in cell fractions contalnlng these nudeoproteins (4), their isolation and characterization are of great importance to the elucidation of protein synthesis. Unfortunately, they are extremely unstable, and their instability increases with purification. One of the factors which influence stability is dialyzable; dialysis of resuspended nucleoprotein pellets (from mouse spleen) for only 3 hours results in a 25 per cent loss of the units which give the characteristic ultracentrifuge patterns (3). I t has now been found that dialysates of rat liver cytoplasm have a stabilizing effect on these nucleoproteins, as described below. All procedures except the ultracen-trifugal analyses were carried out in the cold. The pulp from perfused livers of adult male Wistar rats (1) was homogen-ized in a Waring blendor at a speed which would not break the nuclei (5), in 4 to 6 volumes of 0.88 • sucrose (6), distilled water, or veronal-chloride buffer. Unbroken cells, nuclei, and mitochondria were removed by centrifuging at 20,000 g for 30 minutes when sucrose was used or for 15 minutes in water or buffer. The supernatant was dialyzed in 50 ml. lots against an equal volume of 0.79 M sucrose, distilled water, or buffer, for 16 hours at 5°C., on a rocking platform. These preparations will be referred to as sucrose-SF, water-SF, and buffer-SF. The buffer contained 0.05 ~ sodium chloride, 0.05 ~r sodium diethylbarbiturate, and 0.01 ~ diethylbarbituric acid, and had a pH of 8.6 and an ionic strength of 0.10. For the nudeoprotein preparations mitochondria supematant was prepared in 6 volumes of 0.88 M sucrose as described above, shell-frozen in dry ice and alcohol, and stored in a deep-freeze cabinet. As needed, a portion was thawed quickly by immersing the bottle in water at 37 °, transferred to an ice bath, and titrated to pH 8.3 with 0.05 ~ NaOH in 0.79 M sucrose. One-tenth volume of 5 per cent sodium desoxycholate made up in 0.79 M sucrose at pH 8.3 was added to disrupt the microsome structure (4,7), and …