Depletion Of Cultured Human-Fibroblasts Of Pyridoxal 5'-Phosphate - Effect On Activities Of Aspartate-Aminotransferase, Alanine Aminotransferase, And Cystathionine Beta-Synthase

Abstract Human skin fibroblasts were grown in culture medium containing virtually no pyridoxal. Cells cultured under these conditions grew to confluence for several passages without morphologic signs of degeneration and without changes in activity of two control enzymes, hexokinase and lactate dehydrogenase. The pyridoxal 5′-phosphate content of these fibroblasts fell to about 3% of values obtained during growth in pyridoxal-supplemented medium. The effect of such depletion on the activities of three pyridoxal 5′-phosphate-dependent enzymes was assessed during four consecutive passages. Total activity of cystathionine β-synthase and of aspartate aminotransferase in cell extracts fell to a mean of 50% of control values whereas total activity of alanine aminotransferase remained unchanged. Saturation of these enzymes with cofactor differed as well. The ratio of holoenzyme activity to total enzyme activity fell to less than 15% or predepletion values for cystathionine β-synthase and to 60% for aspartate aminotransferase. In contrast, alanine aminotransferase remained completely saturated with cofactor. Maximal saturation of aspartate amino-transferase with pyridoxal 5′-phosphate was achieved when pyridoxal 5′-phosphate-depleted fibroblasts were grown in medium containing as little as 1 ng/ml of pyridoxal, but addition of 10 ng/ml of pyridoxal was required for maximal saturation of cystathionine β-synthase. Maximal intracellular content of pyridoxal 5′-phosphate was achieved only when 100 ng/ml of pyridoxal was added to the growth medium. Interestingly, the activity of pyridoxine kinase remained constant during all depletion and repletion experiments. We conclude that the ability to grow human fibroblasts under these conditions provides a system for the study of apoenzyme-coenzyme interactions both in intact cultured cells and in cell extracts.