Pyrroline-5-carboxylate synthase activity in mammalian cells.

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Abstract
Although glutamic acid is a precursor for proline biosynthesis, the enzymatic conversion of glutamic acid to pyrroline-5-carboxylic acid (P5C), the immediate precursor of proline, has not been demonstrated in cell-free systems. By providing appropriate concentrations of ATP and NADPH and blocking further metabolism of P5C, a method was developed for measuring the formation of P5C from glutamic acid in homogenates of mammalian cells. This activity is designated P5C synthase. To confirm that the assay is a valid measure of the initial step in proline biosynthesis from glutamic acid, 2 mutant lines of Chinese hamster ovary cells were compared. Proline prototrophic cells, which can synthesize proline from glutamic acid, have easily measurable P5C synthase activity (5.97 nmol P5C/h per mg of homogenate protein). In contrast, proline auxotrophic cells, which are unable to synthesize proline from glutamic acid, have no detectable P5C synthase activity.