Structural analogues of pyrroline 5-carboxylate specifically inhibit its uptake into cells.
Pyrroline 5-carboxylate, a naturally occurring intermediate, is a potent activator of redox-dependent metabolic pathways. The effect of pyrroline 5-carboxylate is due, at least in part, to the special mechanism mediating its entry into cells. Using Chinese hamster ovary cells we recently characterized the cellular uptake of pyrroline 5-carboxylate as a process transferring oxidizing potential pari passu with cell entry, a process consistent with group translocation. We sought to identify specific inhibitors to probe this unique uptake mechanism, to blockade the metabolic effects of pyrroline 5-carboxylate, and to provide strategies to identify the putative carrier protein. Because pyrroline 5-carboxylate, a ring structure with a tertiary nitrogen, is in spontaneous equilibrium with glutamic-gamma-semialdehyde, an open-chain structure, we tested analogues of both. Most open-chain aldehydes at 10 mM had little effect on the uptake of pyrroline 5-carboxylate. Although succinic semialdehyde did inhibit, its effect was nonspecific in that the uptake of alpha(methylamino) isobutyric acid was inhibited as much as the uptake of pyrroline 5-carboxylate. In contrast, pyrroline 2-carboxylate and other cyclic compounds with tertiary nitrogens, e.g., pyridines, were specific inhibitors of pyrroline 5-carboxylate uptake. Respective potencies of pyridine derivatives depended on the nature and location of constituent groups. Kinetics studies showed that these inhibitors were competitive with pyrroline 5-carboxylate and the most potent inhibitor, 2,6-pyridinedicarboxaldehyde, exhibited a K12 of 0.27 +/- 0.05 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
Mixson
A J
AJ
Endocrinology Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Phang
J M
JM
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D001894:Borohydrides / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D007700:Kinetics; D011758:Pyrroles / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D001894:Borohydrides; D002352:Carrier Proteins; D011758:Pyrroles; D013439:Sulfhydryl Reagents; C015485:delta-1-pyrroline-5-carboxylate; C009282:sodium cyanoborohydride