Abstract
Direct evidence is presented for a proline cycle using a cell-free experimental system which sequentially transfers 3H from [1- 3H]glucose to NADP + to Δ 1-pyrroline-5-carboxylate and yields [ 3H]proline. The formation of [ 3H]proline depends on the presence of NADP, Δ 1-pyrroline-5-carboxylate, and the enzymes glucose-6-phosphate dehydrogenase and Δ 1-pyrroline-5-carboxylate reductase. The production of [ 3H]proline from unlabeled proline in the presence of mitochondria provides direct evidence for one complete turn of a proline cycle which transfers reducing equivalents produced by glucose oxidation in the pentose pathway into mitochondria. In this cycle, proline is oxidized to Δ 1-pyrroline-5-carboxylate by mitochondrial proline oxidase. Δ 1-pyrroline-5-carboxylate is released from mitochondria and is recycled back to proline by Δ 1-pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH. At the maximal rate observed, 60% of Δ 1-pyrroline-5-carboxylate produced is recycled back to proline. This cycle provides a mechanism for transferring reducing equivalents from NADPH into mitochondria and is linked to glucose oxidation in the pentose pathway by NADPH turnover.
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