Mutant cell lines resistant to azetidine carboxylic acid: Quantitative and qualitative differences in pyrroline-5-carboxylate synthase activity

Mutant Chinese hamster lung fibroblasts were selected that are resistant to the proline analog L-azetidine-2-carboxylic acid. Resistance in the two mutant cell lines is associated with two distinct alterations in pyrroline-5-carboxylate synthase, the enzyme that catalyzes the proline biosynthetic step leading from glutamic acid to pyrroline-5-carboxylate. In one mutant cell line, pyrroline-5-carboxylate synthase specific activity is increased 30-fold over the level in control cells. In the other mutant line, pyrroline-5-carboxylate synthase activity is not increased, but the enzyme has become insensitive to inhibition by ornithine and proline.     

Mutant cell lines resistant to azetidine-2-carboxylic acid: alterations in the synthesis of proline from glutamic acid

Two mutant Chinese hamster lung fibroblast lines have been isolated that are resistant to the toxic proline analog L-azetidine-2-carboxylic acid. The line designated AZCA-1 has 30-fold elevated activity of pyrroline-5-carboxylate synthase and a large increase in the rate of proline production and release compared to controls. Pyrroline-5-carboxylate synthase activity is not elevated in the resistant line designated AZCA-4, but the enzyme is less sensitive to inhibition by ornithine and proline than control enzyme. Intracellular proline is elevated in AZCA-4 cells, with no change in the rate of release of proline synthesized from glutamate. Resistance to azetidine carboxylic acid in both mutant lines is attributed to the expanded intracellular proline pool that results from alterations in pyrroline-5-carboxylate synthase. These results indicate that intracellular proline levels are determined at least in part by the regulated activity of pyrroline-5-carboxylate synthase.     

Isolation and preliminary characterization of Saccharomyces cerevisiae proline auxotrophs.

Proline-requiring mutants of Saccharomyces cerevisiae were isolated. Each mutation is recessive and is inherited as expected for a single nuclear gene. Three complementation groups cold be defined which are believed to correspond to mutations in the three genes (pro1, pro2, and pro3) coding for the three enzymes of the pathway. Mutants defective in the pro1 and pro2 genes can be satisfied by arginine or ornithine as well as proline. This suggests that the blocks are in steps leading to glutamate semialdehyde, either in glutamyl kinase or glutamyl phosphate reductase. A pro3 mutant has been shown by enzyme assay to be deficient in delta 1-pyrroline-5-carboxylate reductase which converts pyrroline-5-carboxylate to proline. A unique feature of yeast proline auxotrophs is their failure to grown on the rich medium, yeast extract-peptone-glucose. This failure is not understood at present, although it accounts for the absence of proline auxotrophs in previous screening for amino acid auxotrophy.     

Functional Specialization in Proline Biosynthesis of Melanoma

Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of ¹³C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.     

Cloning human pyrroline-5-carboxylate reductase cDNA by complementation in Saccharomyces cerevisiae.

Pyrroline-5-carboxylate reductase (EC 1.5.1.2) catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate to proline. We cloned a human pyrroline-5-carboxylate reductase cDNA by complementation of proline auxotrophy in a Saccharomyces cerevisiae mutant strain, DT1100. Using a HepG2 cDNA library in a yeast expression vector, we screened 10(5) transformants, two of which gained proline prototrophy. The plasmids in both contained similar 1.8-kilobase inserts, which when reintroduced into strain DT1100, conferred proline prototrophy. The pyrroline-5-carboxylate reductase activity in these prototrophs was 1-3% that of wild type yeast, in contrast to the activity in strain DT1100 which was undetectable. The 1810-base pair pyrroline-5-carboxylate reductase cDNA hybridizes to a 1.85-kilobase mRNA in samples from human cell lines and predicts a 319-amino acid, 33.4-kDa protein. The derived amino acid sequence is 32% identical with that of S. cerevisiae. By genomic DNA hybridization analysis, the human reductase appears to be encoded by a single copy gene which maps to chromosome 17.     

Elevated Accumulation of Proline in NaCl-Adapted Tobacco Cells Is Not Due to Altered Delta-Pyrroline-5-Carboxylate Reductase

Tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that are adapted to 428 millimolar NaCl accumulate proline mainly due to increased synthesis from glutamate. These cells were used to evaluate the possible role of Δ¹-pyrroline-5-carboxylate reductase in the regulation of proline biosynthesis. No increase in the specific activity of Δ¹-pyrroline-5-carboxylate reductase in crude extracts throughout the growth cycle was observed in NaCl-adapted cells compared to unadapted cells. The enzyme from both cell types was purified extensively. On the basis of affinity for the substrates NADPH, NADH, and Δ¹-pyrroline-5-carboxylate, pH profiles, chromatographic behavior during purification, and electrophoretic mobility of the native enzyme, the activities of the enzyme from the two sources were similar. These data suggest that the NaCl-dependent regulation of proline synthesis in tobacco cells does not involve induction of pyrroline-5-carboxylate isozymes or changes in its kinetic properties.     

Purified human erythrocyte pyrroline-5-carboxylate reductase. Preferential oxidation of NADPH

Pyrroline-5-carboxylate reductase catalyzes the final step in proline synthesis by NAD(P)H-dependent reduction of pyrroline-5-carboxylate. We have purified and characterized this enzyme from human erythrocytes. Purification to homogeneity (approximately 600,000-fold) was accomplished by sonication, ultracentrifugation, 2',5'-ADP-Sepharose affinity chromatography, and DEAE-Sephacel ion exchange chromatography. The enzyme runs as a single band of 30,000 Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sizing chromatography under nondenaturating conditions demonstrates activity in the 300,000-350,000 Mr range, suggesting that the native enzyme exists as a 10- to 12-mer. The purified enzyme exhibits kinetic characteristics similar to those previously described for whole red cell homogenates. The Vmax is 10-fold higher and the Km for pyrroline-5-carboxylate is 7-fold higher with NADH versus NADPH as cofactor. The affinity for NADPH is 15-fold higher than that for NADH. Erythrocyte pyrroline-5-carboxylate reductase is competitively inhibited by NADP+. Unlike the enzyme from some other sources, erythrocyte pyrroline-5-carboxylate reductase is not inhibited by proline or ATP. Double label studies using [14C]pyrroline-5-carboxylate and [3H]exNADPH in the presence of both NADH and NADPH were performed to determine the preferred source of reducing equivalents. In the presence of physiologic concentrations of pyrroline-5-carboxylate and both pyridine nucleotides, all of the reducing equivalents came from NADPH. We suggest that, in some cell types including human erythrocytes, a physiologic function of pyrroline-5-carboxylate reductase is the generation of NADP+.     

The stimulation of purine nucleotide production by pyrroline-5-carboxylic acid in human erythrocytes

We previously reported that pyrroline-5-carboxylate (PC), the intermediate in the interconversions of proline, ornithine and glutamate markedly stimulates hexosemonophosphate-pentose pathway activity in human erythrocytes. The stimulation is mediated by pyrroline-5-carboxylate reductase which generates NADP⁺ accompanying the conversion of pyrroline-5-carboxylate to proline. We now report that the previously demonstrated effect of pyrroline-5-carboxylate on glucose oxidation through the hexose-monophosphate-pentose pathway is accompanied by increased phosphoribosyl-pyrophosphate production and increased formation of nucleotides via the salvage pathway. The demonstrated effect of pyrroline-5-carboxylate on purine processing may provide a regulatory link between amino acid and nucleotide metabolism.     

Enzymes of ornithine metabolism in adult and developing rat intestine.

The levels of 11 enzymes, most of them involved in the metabolism of ornithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, ornithine aminotransferase, and ornithine transcarbamylase were compared with those in liver. Changes with age (late gestation of adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggest that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotransferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue. Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the ornithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

Metabolism of arginine in lactating rat mammary gland.

Significant activities of the four enzymes needed to convert arginine into proline and glutamate (arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and pyrroline-5-carboxylate dehydrogenase) develop co-ordinately in lactating rat mammary glands in proportion to the increased production of milk. No enzymes were detected to carry out the reactions of proline oxidation or reduction of glutamate to pyrroline-5-carboxylate. Minces of the gland converted ornithine into proline and into glutamate plus glutamine. These conversions increased during the cycle of lactation in proportion to the increased milk production and to the content of the necessary enzymes. The minced gland did not convert labelled ornithine into citrulline, confirming the absence from the gland of a functioning urea cycle, and did not convert labelled proline or glutamate into ornithine. A metabolic flow of labelled arginine to proline and glutamate in mammary gland was confirmed in intact animals with experiments during which the specific radioactivity of proline in plasma remained below that of the proline being formed from labelled arginine within the gland. It was concluded that arginase in this tissue had a metabolic role in the biosynthesis of extra proline and glutamate needed for synthesis of milk proteins.     

A 69 bp fragment in the pyrroline-5-carboxylate reductase promoter of Arabidopsis thaliana activates minimal CaMV 35S promoter in a tissue-specific manner.

The Arabidopsis thaliana gene that encodes pyrroline-5-carboxylate reductase (At-P5R), the last enzyme in proline biosynthesis in A. thaliana, is developmentally regulated and is highly expressed in cells that divide rapidly or undergo changes in osmotic potential. A 69 bp region (P69; -120 to -51) has previously been identified in a 5' deletion analysis of the At-P5R promoter to be necessary for the basal expression. Here, the essential role of P69 for tissue-specific expression of At-P5R is demonstrated by loss- and gain-of-function experiments.     

Enzymes of orithine metabolism in adult developing rat intestine

The levels of 11 enzymes, most of them involved in the metabolism of orithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, orithine aminotransferase, and orithine transcarbamylase were compared with those in liver. Changes with age (late gestation to adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described.The results suggests that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotraferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue.Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the orithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

A radioisotopic assay for delta 1-pyrroline-5-carboxylate synthase activity

A radioisotopic assay is described for measuring the activity of delta 1-pyrroline-5-carboxylate synthase, the enzyme that catalyzes the formation of delta 1-pyrroline-5-carboxylic acid from glutamic acid. Pyrroline-5-carboxylic acid is a common intermediate in the pathways through which glutamic acid, proline, and ornithine are interconverted. To determine pyrroline-5-carboxylate synthase activity, cell homogenates are incubated with [14C]-glutamic acid, the products of the reaction are converted quantitatively to proline by sodium borohydride, and proline is isolated by cation-exchange column chromatography. Cofactor requirements have been defined, and the activity of pyrroline-5-carboxylate synthase in several different cultured fibroblast lines is reported.     

Pyrroline-5-carboxylate synthesis from glutamate by rat intestinal mucosa

The mitochondria of rat intestinal mucosa were found to have an enzymatic activity that converts radioactive glutamate to pyrroline-5-carboxylate (P5C) in the presence of ATP, NADPH, and MgCl2. The product of this enzyme was identified as P5C by the fact that it was converted to proline by chemical reduction with NaBH4 or by enzymatic reduction with NADH in the presence of purified yeast P5C reductase. The product was demonstrated to be P5C rather than pyrroline-2-carboxylate by thin layer chromatography. The presence of the activity in mitochondria prepared from intestinal mucosa of germ-free rats proved that this activity is of mammalian origin. Omission of either ATP, NADPH, or MgCl2 from the reaction mixture resulted in little or no activity. The optimal pH appeared to be about 7.0 under the conditions used. Substrate saturation curves in the presence of an ATP and an NADPH regeneration system gave apparent Km values of 2.5 mM for glutamate, 0.19 mM for ATP, and 6.5 microM for NADPH in the presence of 20 mM MgCl2. The mitochondrial preparation usually produced P5C at a rate of 1.2 to 1.6 nmol/mg/min at 20 degrees C when incubated with 1 mM glutamate, 3 mM ATP, 0.2 mM NADPH, and 20 mM MgCl2.     

Proline dehydrogenase and pyrroline-5-carboxylate reductase from pumpkin cotyledons

Activity of proline dehydrogenase and pyrroline-5-carboxylate reductase was greatest after 5 and 7 days germination in green and etiolated cotyledons respectively of pumpkin (Cucurbita moschata Poir. cv. Dickinson Field). The ratio of pyrroline-5-carboxylate reductase to proline dehydrogenase activity was constant throughout germination. Both enzymes were purified 30-fold but the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase activity was constant throughout purification. However, this ratio decreased with storage, especially in purified preparations. Both enzymes were stable at high temperature and the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase remained unchanged on heating. Proline dehydrogenase and pyrroline-5-carboxylate reductase were inhibited by sodium bisulfite and cysteine. ATP, ADP and NADP caused inhibition of both enzymes. Proline dehydrogenase utilized NAD but not NADP. Pyrroline-5-carboxylate reductase had a 2.5-fold greater activity with NADH than NADPH. Most of the data presented suggest that proline dehydrogenase and pyrroline-5-carboxylate reductase activities occur on the same protein molecule.     

Alterations in the activities of the enzymes of proline metabolism in Ragi (Eleusine coracana) leaves during water stress

Free proline content in Ragi (Eleusine coracana) leaves increased markedly (6 to 85 fold) as the degree of water stress, created by polyethylene gylcol treatment, was prolonged There was also a marginal increase in soluble proteins in the stressed leaves as compared to that in the controls. Water stress stimulated the activities of ornithine aminotransferase and pyrroline-5-carboxylate reductase, the enzymes of proline biosynthesis and markedly inhibited the enzymes involved in proline degradation viz., proline oxidase and pyrroline-5-carboxylate dehydrogenase. These results suggest that increase in free proline content of Ragi leaves could be due to enhanced activities of the enzymes synthesizing proline but more importantly due to severe inhibition of the enzymes degrading proline. These observations establish for the first time, the pathway of proline metabolism in plants by way of detection of the activities of all the enzymes involved and also highlight the role of these enzymes in proline accumulation during water stress.     

Activities of the pentose phosphate pathway and enzymes of proline metabolism in legume root nodules

Based on localization and high activities of pyrroline-5-carboxylate reductase and proline dehydrogenase activities in soybean nodules, we previously suggested two major roles for pyrroline-5-carboxylate reductase in addition to the production of the considerable quantity of proline needed for biosynthesis; namely, transfer of energy to the location of biological N₂ fixation, and production of NADP⁺ to drive the pentose phosphate pathway. The latter produces ribose-5-phosphate which can be used in de novo purine synthesis required for synthesis of ureides, the major form in which biologically fixed N₂ is transported from soybean root nodules to the plant shoot. In this paper, we report rapid induction (in soybean nodules) and exceptionally high activities (in nodules of eight species of N₂-fixing plants) of pentose phosphate pathway and pyrroline-5-carboxylate reductase. There was a marked increase in proline dehydrogenase activity during soybean (Glycine max) ontogeny. The magnitude of proline dehydrogenase activity in bacteroids of soybean nodules was sufficiently high during most of the time course to supply a significant fraction of the energy requirement for N₂ fixation. Proline dehydrogenase activity in bacteroids from nodules of other species was also high. These observations support the above hypothesis. However, comparison of pentose phosphate pathway and pyrroline-5-carboxylate reductase activities of ureide versus amide-exporting nodules offers no support. The hypothesis predicts that pyrroline-5-carboxylate and pentose phosphate pathway activities should be higher in ureide-exporting nodules than in amide-exporting nodules. This predicted distinction was not observed in the results of in vitro assays of these activities.     

Purification to homogeneity of pyrroline-5-carboxylate reductase of barley

An enzyme has been purified to homogeneity from barley seedlings which has `proline dehydrogenase' and the pyrroline-5-carboxylic acid reductase activities. The purification achieved is 39,000-fold as calculated from the proline dehydrogenase activity. The subunit molecular weight of the protein is 30 kilodaltons. The native enzyme has molecular weights up to 480 kilodaltons, depending on the buffer environment. From the pH profiles, the specific activities and thermodynamic considerations, it is concluded that the plant proline dehydrogenase functions in vivo as a pyrroline-5-carboxylate reductase.     

Stimulation of the hexose-monophosphate pentose pathway by delta 1-pyrroline-5-carboxylic acid in human fibroblasts.

L-pyrroline-5-carboxylic acid, an intermediate in the interconversions of glutamic acid, ornithine and proline, is a potent stimulator of the hexose-monophosphate pentose pathway in cultured human fibroblasts. These studies suggest that pyrroline-5-carboxylate reductase, which catalyzes the conversion of pyrroline-5-carboxylate to proline coupled with the oxidation of NADPH, provides the NADP for the observed activation of the hexose-monophosphate pentose pathway.     

Genetic analysis of Pycr1 and Pycr2 in mice

The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to proline. Mutations in human PYCR1 and ALDH18A1 (P5C Synthetase) cause Cutis Laxa (CL), whereas mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10). Here, we investigated the genetics of Pycr1 and Pycr2 in mice. A null allele of Pycr1 did not show integument or CL-related phenotypes. We also studied a novel chemically-induced mutation in Pycr2. Mice with recessive loss-of-function mutations in Pycr2 showed phenotypes consistent with neurological and neuromuscular disorders, including weight loss, kyphosis, and hind-limb clasping. The peripheral nervous system was largely unaffected, with only mild axonal atrophy in peripheral nerves. A severe loss of subcutaneous fat in Pycr2 mutant mice is reminiscent of a CL-like phenotype, but primary features such as elastin abnormalities were not observed. Aged Pycr2 mutant mice had reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder. PYCR1 and -2 have similar enzymatic and cellular activities, and consistent with previous studies, both were localized in the mitochondria in fibroblasts. Both PYCR1 and -2 were able to complement the loss of Pro3, the yeast enzyme that converts P5C to proline, confirming their activity as P5C reductases. In mice, Pycr1; Pycr2 double mutants were sub-viable and unhealthy compared to either single mutant, indicating the genes are largely functionally redundant. Proline levels were not reduced, and precursors were not increased in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, but placing Pycr2 mutant mice on a proline-free diet worsened the phenotype. Thus, Pycr1 and -2 have redundant functions in proline biosynthesis, and their loss makes proline a semi-essential amino acid. These findings have implications for understanding the genetics of CL and HLD10, and for modeling these disorders in mice.