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Δ1-pyrroline-5-carboxylate reductase ( l -proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) from Baker's yeast has been purified and characterized. Purification to an apparently homogenous protein was effected by using ‘reagent-grade’ water containing dithiothreitol and by maintaining a constant pH 7.5, because of the instability of the enzyme protein. The enzyme was purified approximately 200-fold from the crude extract of baker's yeast, and it is a negatively-charged protein with a molecular weight of 125 000, containing an active SH group which participates in binding with NAD(P)H. The Km value for dl -pyrroline-5-carboxylate is 0.8 · 10−4 M; for NADH, it is 4.8 · 10−5 M; and for NADPH, it is 5.6 · 10−5 M. These Km values are much smaller than those of enzymes from other sources. The purified enzyme is free of contaminating enzymes which might interfere with its use in assays. The enzyme has been applied successfully to the assays of l Δ 1 - pyrroline -5- carboxylate and l -ornithine in tissue, and in vivo levels of these amino acids in rat liver are reported.

A homogeneous preparation of Δ1-pyrroline-5-carboxylate reductase ( l -proline: NAD (P)+ 5-oxidoreductase, EC 1.5.1.2) was obtained from baker's yeast by an affinity chromatography, using 5′ AMP-Sepharose 4B. After the 1st DEAE-Sephadex column chromatography, the enzyme absorbed on 5′ AMP-Sepharose column was eluted with 1 mM ATP. The final preparation was homogeneous on polyacrylamide gel electrophoresis and purified 3500-fold from the crude extract. This purified enzyme was very useful as a coupling enzyme for the assays of ornithine and pyrroline-5-carboxylate in tissues, and for the rate assay of ornithine aminotransferase activity.