THE DYNAMICS OF COBALAMIN UTILIZATION IN L-1210 MOUSE LEUKEMIA-CELLS - A MODEL OF CELLULAR COBALAMIN METABOLISM

The uptake and metabolism of cobalamin (Cbl) has been studied in L-121 0 murine leukemia cells propagating in vitro. Extracellular Cbl (prote in bound and free) and intracellular Cbl (protein bound and free) were determined after culturing L-1210 cells in the presence of [Co-57]cya nocobalamin (CN-Cbl) bound to transcobalamin II (transcobalamin, TC). The intracellular pool of free [Co-57]Cbl increased during the first 2 4 h of culture and a substantial fraction of this free pool was efflux ed from the cell to the medium. Upon depletion of extracellular TC-[Co -57]CN-Cbl, the intracellular concentration of free Cbl decreased as d id the efflux of Cbl to the medium. Internalized [Co-57]CN-Cbl was con verted to hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl) and 5'-d eoxyadenosylcobalamin. These Cbl forms were found in both soluble (cyt oplasmic) and insoluble (membrane) fractions. Intracellular protein-bo und [Co-57]Cbl fractionated with methionine synthase (MS) and methylma lonyl-CoA mutase (MU) activity. The major form of Cbl associated with the two enzymes was OH-Cbl. Cells propagated in medium containing N-5- methyltetrahydrofolate and homocysteine showed a substantial increase in MS activity which paralleled the increase in the intracellular conc entration of Me-Cbl and the Cbl bound to the enzyme.