Effects of the Proline Analog l-Thiazolidine-4-carboxylic Acid on Proline Metabolism

Abstract

The effect of various proline analogs on proline oxidation in mitochondria isolated from etiolated barley (Hordeum vulgare) shoots was investigated. Ofthe analogs tested, only L-thiazolidine-4-carboxylic acid (T4C) was an effective inhibitor. T4C (1 millimolar) inhibited proline (10 millimolar) -dependent 02 uptake an average of 67%. T4C was also oxidized to some degree (12.9 nanoatoms oxygen per minute per milligram protein for 10 millimolar). The effect of T4C on the oxidation of other mitochondrial substrates was also tested. T4C inhibited A'-pyrrolidine-5-carboxylic acid-dependent oxygen uptake slightly (13%), the oxidation of malate plus pyruvate even less (6%), and stimulated the oxidation of succinate (+11%), exogenous NADH (+19%), and citrate (+20%). Thus, inhibition by T4C in mitochondria is relatively specific to proline oxidation. T4C was found to inhibit proline dehydrogenase and not the transport of proline into the matrix.The effect of T4C on proline metabolism in detached green barley leaves was investigated. T4C inhibited proline oxidation in turgid leaves, increasing the proline content of these leaves slightly. In wilted leaves (that are synthesizing proline rapidly), T4C inhibited proline synthesis, which resulted in a decrease in the proline content of the leaves. A'-pyrrolidine-5-arboxylic acid reductase (the last enzyme in proline synthesis) was not inhibited by T4C, and thus T4C's influence is prior to that step of the synthetic pathway. T4C had no influence on the incorporation of proline into protein.genases) are bound to the matrix side of the inner mitochondrial membrane where they are associated with the first portion of the electron transport chain (7,8). Therefore, proline must penetrate the inner mitochondrial membrane for oxidation to occur. The fact that proline uptake into the matrix is stereospecific (5), reversibly sensitive to sulfhydryl reagents (5), and sensitive to uncouplers (8) suggests that proline uptake is a carrier-mediated energy-dependent process.The oxidation of proline is more sensitive to water stress than is the oxidation of other mitochondrial substrates (18). Since proline oxidation can be envisioned as being regulated at either the transport or dehydrogenase level, a proline oxidation inhibitor would be very useful in investigating the integration and regulation of these two mitochondrial processes. Such an inhibitor would also be useful in further investigating the cellular relationships that lead to proline accumulation during environmental stress, and for assessing the role of proline oxidation in providing energy and carbon skeletons for recovery after stress. A proline oxidation inhibitor would be especially useful for investigating pollen and spore germination in species where most of the required energy is believed to be derived from proline oxidation.In this paper, we present evidence that T4C is a relatively specific inhibitor of proline oxidation in barley mitochondria. We have also looked at T4C's effect on proline levels and on proline me...

Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation

“…Different nucleotide specificities have also been reported for varieties of the same species; while P5C reductase from Hordeum vulgare var Augusta was most active with NADPH (9), the enzyme from H. vulgare var Larker preferred NADH over NADPH by a factor of at least threefold (4). Some of this discrepancy could be explained if two P5C reductases were present in plants as has already been suggested (4,17). However, no isozymes of P5C reductase were found in C. autotrophica or C. saccharophila.…”

“…More recently, Treichel (23) showed that P5C reductase activity and substrate affinity increased with progressive adaptation to NaCl stress of cell suspension cultures of Mesembryanthemum nodiflorum. Cells grown in 400 mM NaCl showed nearly 4 times more activity of P5C reductase than cells cultivated 'Research supported by grant A6032 from the Natural Sciences and Engineering Research Council of Canada. 2Abbreviations: P5C, Av-pyrroline-5-carboxylate; ASW, artificial sea water; FPLC, fast protein liquid chromatography. 917 in the absence of NaCl.…”

Proline Dehydrogenase Contributes to Pathogen Defense in Arabidopsis

“…Extracts were centri-fuged at 16,000g for 10 min, and supernatants were used to determine ProDH activity in vitro by incubation in 0.15 M Na 2 CO 3 -HCl buffer (pH 10.3) with 1 mM NAD + and 20 mM L-Pro to monitor the NAD + reduction at 340 nm. The specificity of the enzymatic reaction was evaluated by incubation of samples with the ProDH inhibitor L-4-thiazolidine carboxylic acid (Elthon and Stewart, 1984 …”

Oxidation of L-thiazolidine-4-carboxylate by L-proline dehydrogenase in Escherichia coli

“…In previous studies (Mackenzie & Harris, 1957;Unger & DeMoss, 19663;Elthon & Stewart, 1984), oxidation of T4C by intact bacteria or mitochondria was measured manometrically as oxygen uptake. To determine if T4C degradation in E. coli is mediated by the membraneassociated enzyme proline dehydrogenase, a spectrophotometric assay for T4C catabolism was developed.…”