Spectrophotometric measurement of pyruvate dehydrogenase complex activity in cultured human fibroblasts

A spectrophotometric assay for the pyruvate dehydrogenase complex (PDHC) has been adapted for use with cultured human fibroblasts. It is a coupled enzyme assay utilizing pigeon liver arylamine acetyltransferase to measure the acetyl-CoA produced by PDHC. Activity is proportional to fibroblast protein and to time and depends completely on added pyruvate, CoA and NAD. In extracts in which PDHC had been activated (dephosphorylated) by the method of Sheu et al. (Sheu, R.K.-F., Hu, C.C. and Utter, M.F. (1981) J. Clin. Invest. 67, 1463-1471), activities in control cell lines are 5-50-fold higher than in earlier reports. Low activity has been demonstrated in a line previously reported to be PDHC-deficient.