Biosynthesis of transcobalamin II by adult rat liver parenchymal cells in culture

The biosynthesis of transcobalamin II was investigated in primary cultures of adult rat liver parenchymal cells maintained in serum-free media. The data indicate that these hepatocytes secrete a vitamin B 12-binding substance into the culture medium which is identical to rat serum transcobalamin II as judged by the following criteria: (i) gel filtration on columns of Sephadex G-200; (ii) ion-exchange chromatography on columns of diethyl aminoethyl cellulose and carboxymethyl cellulose; (iii) polyacrylamide-gel electrophoresis at pH 9.5; and (iv) the ability to facilitate cellular vitamin B 12 uptake by HeLa cells and mouse L-929 fibroblasts in culture. The secretion of transcobalamin II by the liver parenchymal cells was blocked by cycloheximide, puromycin, and p-fluorophenylalanine. The inhibition by cycloheximide, but not that of the other inhibitors, was partially reversed upon removal of the drug. The liver parenchymal cells incorporated radioactive amino acids into transcobalamin II which was absorbed from the growth medium using affinity chromatography on Sepharose containing covalently linked B 12. Collectively, these data indicate that rat liver parenchymal cells, in culture, are capable of the biosynthesis de novo of transcobalamin II and the subsequent secretion of this protein into the culture media.