Protein Synthesis in Single Cell Suspensions from Rat Liver

Suspensions of single cells were prepared from normal adult rat liver by perfusion of liver with and shaking in buffered solutions of 0.1% hyaluronidase and 0.05% collagenase. l -[1-14C]Leucine and l -[1-14C]methionine incorporated into protein could be measured by precipitating the cells on filter paper disks without prior homogenization. No incorporation of amino acids into protein was observed if the cells were incubated under nitrogen. Optimal incorporation was obtained in 3 m m to 12 m m phosphate buffer at pH 7.17. The incorporation was inhibited at higher phosphate concentrations, and it was also less efficient in Tris-HCl buffer, Tris-maleate buffer, and Tris-phosphoric acid buffer. The incorporation of l -[1-14C]leucine into protein was directly proportional to the concentration of the cells in the suspension. High doses of l -leucine or l -methionine (∼2 m m ) did not inhibit the incorporation of amino acids into protein. The cells were freely permeable for l -leucine and l -methionine. The system incorporating amino acids into protein was half-saturated with 7.2 x 10-5 m l -leucine and 5.1 x 10-5 m l -methionine. An inhibition of 50% of the incorporation of l -leucine or l -methionine into protein was observed at a puromycin concentration of 1.4 µg per ml, or 0.8 µg per ml, respectively. Changing the incubation temperature from 22° to 32° caused a 4-fold increase of the initial rate of incorporation of l -leucine or l -methionine.