Branched-chain ketoacid dehydrogenase activity and growth of normal and mutant human fibroblasts: the effect of branched-chain amino acid concentration in culture medium.

Abstract

We investigated changes in cell growth and branched chain ketoacid dehydrogenase (BCKD) activity by varying the concentrations of branched-chain amino acids (BCAAs) in culture medium of diploid fibroblasts from humans with normal BCKD and with impaired enzyme function. For logarithmic growth the two cell populations required similar minimal concentrations (0.05 mM) for each of leucine, isoleucine, and valine tested together. At confluency (saturation density) mutant cells grew less well to the extent of 30 to 40% in the highest concentrations of BCAAs that could be tested, 20.8 mM. BCKD activity was not changed by growth of normal or mutant cells in the absence of BCAAs. This enzyme activity was increased in normal but not mutant cells by growth in 20.8 mM BCAAs. These studies suggest the following: (1) BCKD mutant fibroblasts in culture slow their growth rate in response to high concentrations of BCAAs; (2) the growth disadvantage for mutant cells in high concentrations of BCAAs may be useful to select for enzyme normal hybrids derived when cells with two different mutations affecting BCKD are fused; (3) the increase of BCKD activity in normal but not mutant cells grown in high concentrations of BCAAs can distinguish these phenotypes more precisely in humans; and (4) the mechanism of BCKD stimulation in normal cells grown in high concentrations of BCAAs remains to be explained but can be pursued further with the cell culture conditions described.