| Abstract: | Abstract Pyrroline 5-carboxylate, a potent effector of redox dependent pathways, is a nutritionally responsive plasma constituent and is present in tissue culture medium conditioned by a variety of cells. Studies of P5C production and release both in vivo and in vitro have become increasingly important. To date, the assay used is a specific, sensitive radioenzymatic method. Unfortunately, the necessary materials (purified P5C reductase and NADP3H], labeled in the transferable position) are not readily available. We now describe a method using commercially available reactants. This new method, based on the interaction of P5C with cysteine, is sensitive and specific; the presence of a wide variety of compounds only minimally affected the assay. S35] Cysteine at a defined specific activity is reacted with P5C and the P5C-CYS adduct can be separated from the reactants by cation exchange column chromatography. To optimize the assay conditions, we characterized the time course, pH dependence and cysteine concentrations for adduct formation. Studies showed that the sulfhydryl group of cysteine is critical in the formation of the P5C-CYS adduct. Comparison of this new method to the previously published radioenzymatic method showed close correlation over a wide range of P5C concentrations in conditioned media. |
