Demonstration of a NADPH-linked Δ1-pyrroline-5-carboxylate-proline shuttle in a cell-free rat liver system

Abstract

These studies indicate that the interconversions of Δ 1-pyrroline-5-carboxylate and proline can function as a shuttle that generates extra-mitochondrial NADP + and transfers hydride ions into mitochondria in a cell-free rat liver system. A phosphate-free buffer with high concentrations of triethanolamine and 2-mercaptoethanol prevented the cold inactivation of pyrroline-5-carboxylate reductase (ED 1.5.1.2) in liver extracts. This enzyme had an apparent K m NADPH that was 2% of the apparent K m NADH. V max NADPH was approx. 50% of V max NADH. Unlabeled proline was converted to [5- 3H]proline in incubations containing liver soluble fraction, mitochondria and a [4 S- 3H]NADPH generating system. This demonstrated one turn of the proposed shuttle in a homologous liver system. [5- 3H]Proline production increased linearly over 60 min and decreased by 87% or more when specific components were eliminated. Rotenone was required for maximal activity, suggesting that inhibition of Δ 1-pyrroline-5-carboxylate efflux would be required for significant shuttle activity in vivo. Both the relative concentrations of NADPH and NADH in liver cytosol and the kinetic characteristics of liver pyrroline-5-carboxylate reductase predict that the described shuttle should be overwhelmingly linked to NADPH rather than NADH. A NADPH-linked Δ 1-pyrroline-5-carboxylate-proline shuttle may occur in hepatocytes and function at specific times to regulate pathways limited by cytosolic [NADP +].