Fig. 3.

Aminotransferase screening assay used to determine the alanine biosynthetic route in human RBCs. Cell lysates were incubated with 200 μM pyridoxal-5′-phosphate, 5 mM pyruvate, and 5 mM of a target amino acid. Each square shows the unique ¹H–¹³C NMR sub-spectrum used to identify and quantify each metabolite observed in the RBC lysates. Row labels denote the compounds added to samples, and column labels denote metabolite signals observed by ¹H–¹³C NMR. In the first row, no amino acid was added in addition to pyruvate; in subsequent rows, the indicated amino acid was added in addition to pyruvate. Abbreviations: Pyr pyruvate, αKG α-ketoglutarate, amino acids are represented by standard three letter code. ^‡The samples prepared with glutamate showed signals from both the substrate (glutamate) and the two products (alanine and α-ketoglutarate). ^†Samples incubated with glutamine showed no detectable levels of GA activity. However, GA activity was detectable by MS