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Activity of proline dehydrogenase and pyrroline-5-carboxylate reductase was greatest after 5 and 7 days germination in green and etiolated cotyledons respectively of pumpkin ( Cucurbita moschata Poir. cv. Dickinson Field). The ratio of pyrroline-5-carboxylate reductase to proline dehydrogenase activity was constant throughout germination. Both enzymes were purified 30-fold but the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase activity was constant throughout purification. However, this ratio decreased with storage, especially in purified preparations. Both enzymes were stable at high temperature and the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase remained unchanged on heating. Proline dehydrogenase and pyrroline-5-carboxylate reductase were inhibited by sodium bisulfite and cysteine. ATP, ADP and NADP caused inhibition of both enzymes. Proline dehydrogenase utilized NAD but not NADP. Pyrroline-5-carboxylate reductase had a 2.5-fold greater activity with NADH than NADPH. Most of the data presented suggest that proline dehydrogenase and pyrroline-5-carboxylate reductase activities occur on the same protein molecule.

The product of oxidation of proline by pumpkin proline dehydrogenase reacted with o -aminobenzaldehyde to give a yellow compound that had an absorption spectrum similar to that obtained from chemically synthesized Δ 1 -pyrroline-5-carboxylate. The product of the proline dehydrogenase reaction and synthetic Δ 1 -pyrroline-5-carboxylate had identical R f values. Both authentic Δ 1 -pyrroline-5-carboxylate and the product of the enzyme gave a pink colour with acid ninhydrin on paper chromatograms and both had identical elution patterns on Dowex 50(H + ) columns. Neither synthetic Δ 1 -pyrroline-5-carboxylate nor the product of proline-dehydrogenase produced γ-amino butyrate with hydrogen peroxide.

A NAD specific proline-dehydrogenase was found in pumpkin (Cucurbita moschata Poir. cv. Dickinson Field) which oxidized proline to Δ1-pyrroline-5-carboxylate. NADP did not substitute for NAD and L-proline-methyl-ester and thiazolidine-4-carboxylate were substrates in the reaction, at a rate of 107% and 33% respectively, of the rate with L-proline. Pumpkin cotyledons contained the bulk of the enzyme activity with 90% of the activity being in the soluble fraction. Proline-dehydrogenase, which was not treated at high temperature, was stable at –10°C for 4 months in the presence of high ammonium sulfate concentration. The Michaelis constant for NAD was 2.2 mM and for L-proline was 2.5 mM. At 5 mM NADP, a 40% non-competitive inhibition of proline-dehydrogenase was obtained, while 50 μM NADP was sufficient to induce 20% inhibition.