Demonstration of a NADPH-linked delta 1-pyrroline-5-carboxylate-proline shuttle in a cell-free rat liver system.
These studies indicate that the interconversions of delta 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 (EC 1.5.1.2) in liver extracts. This enzyme had an apparent KmNADPH that was 2% of the apparent KmNADH X VmaxNADPH was approx. 50% of VmaxNADH. Unlabeled proline was converted to [5-3H]proline in incubations containing liver soluble fraction, mitochondria and a [4S-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 delta 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 delta 1-pyrroline-5-carboxylate-proline shuttle may occur in hepatocytes and function at specific times to regulate pathways limited by cytosolic [NADP+].
D000818:Animals; D002474:Cell-Free System; D003080:Cold Temperature; D008930:Mitochondria, Liver / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010427:Pentose Phosphate Pathway; D011392:Proline / Q000378:metabolism*; D011758:Pyrroles / Q000378:metabolism*; D011762:Pyrroline Carboxylate Reductases / Q000378:metabolism; D051381:Rats; D012995:Solubility
D011758:Pyrroles; C015485:delta-1-pyrroline-5-carboxylate; D009249:NADP; D011392:Proline; D011762:Pyrroline Carboxylate Reductases