Action of a proline analogue, l-thiazolidine-4-carboxylic acid, in Escherichia coli

Unger, Leon (University of Illinois, Urbana), and R. D. DeMoss. Action of a proline analogue, l-thiazolidine-4-carboxylic acid, in Escherichia coli. J. Bacteriol. 91:1556-1563. 1966.-The effect of the proline analogue, l-thiazolidine-4-carboxylic acid (thioproline), on growth, and its relation to the metabolic function of proline in protein synthesis in Escherichia coli K-12, has been studied. Thioproline causes linear growth in E. coli within one generation. The inhibition is specifically reversed by the simultaneous addition of l-proline. Thioproline, or a closely related metabolic derivative, is incorporated into bacterial proteins. Proline antagonizes the incorporation of "thioproline" into protein. The analogue specifically inhibits the rate and extent of prolyl-ribonucleic acid formation. The effectiveness of thioproline as a proline analogue is attributed to its ability to interfere with the utilization of proline for protein synthesis and to mimic proline in its function of being incorporated into proteins. The effect of the incorporation of thioproline on protein structure and enzyme activity is discussed.     

The requirements for bicarbonate and metabolism of the inducer during germ tube formation by Candida albicans

Factors affecting germ tube formation in Candida albicans at suboptimal temperatures were investigated. Candida albicans formed germ tubes between 22 and 30 degrees C in solution when incubated without shaking, in the presence of bicarbonate (2 mg mL-1). Other conditions depended on the inducer used. Proline could induce germ tube formation optimally only when its concentration was between 200 and 400 mM. A concentration of 0.05 mM N-acetylglucosamine was sufficient to induce germ tube formation. N-Acetylglucosamine could induce germ tube formation at 30 but not at 25 degrees C. N-Acetylglucosamine induced germ tube formation was most reproducible when the cells were first starved by incubation in water for 16-24 h at 20 degrees C. Germ tubes induced by proline could be formed at pH values between 3.8 and 9.0 at 30 degrees C, but only between 7.0 and 7.5 at 25 degrees C. The addition of 0.05 to 5 mM glucose to a 5 mM proline induction solution allowed germ tube formation at 30 but not at 25 degrees C. Glucose (400 mM) did not suppress germ tube formation at 30 degrees C but only 5 mM was sufficient to cause a 65% suppression at 25 degrees C. The results show the importance of CO2 and (or) bicarbonate to the induction of germ tube formation and are consistent with the metabolism of the inducer.     

SLC36A4 (hPAT4) is a high affinity amino acid transporter when expressed in Xenopus laevis oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.     

Characteristics of proline transport into R3230AC mammary tumor cells

Cells separated by enzyme treatment of the R3230AC mammary carcinoma were used to characterize the entry of proline. These cells showed minimal changes in cell viability and intracellular volume and were found to be suitable for transport studies, since the vi of proline was maintained for at least 4 h when cells were stored at 37 or 4 degrees C, or when transport was measured in the presence or absence of Na+. Proline was acitvely transported by these tumor cells, reaching a distribution ratio ([proline] intracellular/[proline] extracellular) of 20 after 2 h. Proline entry consisted of two processes, one saturable (carrier mediated) and the other, non-saturable. The carrier-mediated entry, Km - 0.83 mM and V = 151.10(-5) mumol/min per 5.10(6) cells, was Na+-dependent, sensitive to pH and metabolic inhibitors, and completely inhibited by alpha-(methylamino)-isobutyric acid (Ki = 0.34 mM). Proline entry in the absence of Na+ was 20% that in the presence of Na+ and was found to be due to a non-saturable process, since (a) vi of proline uptake in the absence of Na+ increases linearly with increasing proline concentration and (b) was not suppressed by either 20 mM alpha-(methyl-amino)-isobutyric acid, 50 mM glycine +20 mM phenylalanine, or 50 mM serine +20 mM phenylalanine when proline uptake was measured in the presence or absence of Na+. Therefore, under the conditions studied, we conclude that proline transport appears to be restricted to the A (alanine-preferring) system. Furthermore, these cells should provide a suitable model to study the effect of hormonal manipulations on the amino acid transport process.     

Effects of salt and proline on Medicago sativa callus

In this study, two cultivars of Medicago sativa (cv. Yazdi and cv. Hamedani) were used for callus production. Calluses were transferred to MS medium containing 0, 30, 60, 90, and 120 mM NaCl and 0, 5, 10 mM proline. After 4–5 weeks dry weight and intracellular free proline of the calluses were measured. The growth of callus in both cultivars decreased with increasing salt concentration. Addition of exogenous proline to the culture medium increased the dry weight and free proline content of callus. The difference between control and treated calluses with 10 mM exogenous proline in the medium was significant. The data obtained from experiments indicated that the responses of two Medicago cultivars was genotype dependent.     

Metabolism of a proline analogue, l-thiazolidine-4-carboxylic acid, by Escherichia coli

Unger, Leon (University of Illinois, Urbana), and R. D. DeMoss. Metabolism of a proline analogue, l-thiazolidine-4-carboxylic acid, by Escherichia coli. J. Bacteriol. 91:1564-1569. 1966.-Resting cells of Escherichia coli K-12, pregrown in a proline- and thioproline-free medium, oxidize the proline analogue, l-thiazolidine-4-carboxylic acid (l-thioproline), without a lag with the consumption of 1 atom of oxygen per mole of thioproline. The organism also oxidizes cysteine and formaldehyde, the chemical precursors of thioproline. The total oxygen consumed is the same whether the substrate is thioproline, cysteine, formaldehyde, or an equimolar mixture of cysteine and formaldehyde. The results suggest that neither cysteine nor formaldehyde are free intermediates in the oxidative pathway. Thioproline is available as a metabolic carbon source for the synthesis of the ribonucleic acid bases, guanine and uracil.     

The genotoxicities of N-nitrosamines in Drosophila melanogaster in vivo: the correlation of mutagenicity in the wing spot test with the DNA damages detected by the DNA-repair test

The genotoxicities of a series of N-nitrosamines were assayed in the wing spot test and a new short-term test of Drosophila melanogaster. In the spot test, larval flies trans-heterozygous for the somatic cell markers mwh and flr3 were fed the test reagents and the wing hairs in adults were inspected for clones expressing the phenotypes of the markers. In the other test, larval stock consisting of meiotic recombination-deficient (Rec-) double mutant mei-9a and mei-41D5 males and repair-proficient Rec+ females were grown on feed containing the reagents and the DNA damages were detected with the preferential killing of the Rec- larvae as an endpoint. The carcinogenic nitrosamines tested, N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-butylamine (NDBA), N-nitrosomorpholine (NMOR), N-nitro-sopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR), all showed clearly positive activities in both tests. The activities in the wing spot test were ranked in a sequence of NDMA much greater than NMOR greater than NPIP greater than NDEA greater than NPYR greater than NDBA. A similar ranking was obtained in the repair assay. The genotoxicity of N-nitrosodiphenylamine (NDPhA), carcinogenicity studies of which are inconclusive, was marginal in the spot test. The non-carcinogenic N-nitrosoproline (NPRO) and the non-mutagenic N-nitrosothioproline (NTPRO) were negative in the spot test. NDPhA and NPRO were negative in the repair test as well. The DNA-repair test is thus a convenient technique for estimating the mutagenicity of compounds because of its simplicity compared with the wing spot test. These Drosophila tests may be useful in predicting carcinogenic potentials of compounds.     

Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells

We present a fully defined culture system (adapted Essential8^TM [E8^TM] medium in combination with vitronectin) for human embryonic stem cells that can be used for SILAC purposes. Although a complete incorporation of the labels was observed after 4 days in culture, over 90% of precursors showed at least 10% conversion. To reduce this arginine conversion, E8^TM medium was modified by adding (1) l ‐proline, (2) l ‐ornithine, (3) N^ω‐hydroxy‐nor‐l ‐arginine acetate, or by (4) lowering the arginine concentration. Reduction of arginine conversion was best obtained by adding 5 mM l ‐ornithine, followed by 3.5 mM l ‐proline and by lowering the arginine concentration in the medium to 99.5 μM. No major changes in pluripotency and cell amount could be observed for the adapted E8^TM media with ornithine and proline. However, our subsequent ion mobility assisted data‐independent acquisition (high‐definition MS) proteome analysis cautions for ongoing changes in the proteome when aiming at longer term suppression of arginine conversion.     

Embryogenic callus formation,growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda Giganteus' as affected by proline

The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and 22.6 μM 2,4-dichlorophenoxyacetic acid. Shoot apices and leaves from in vitro-propagated shoots, and immature inflorescences from greenhouse-grown plants were used as explants for callus induction and formation. Suspension cultures initiated from embryogenic callus of immature inflorescences were used to test the effect of proline in suspension cultures. The proline additions affected the formation of embryogenic callus and the growth of suspension cultures. Improvements depended on the proline concentration and the basal salts of the medium. Addition of 12.5 to 50 mM proline to callus induction medium with Murashige and Skoog salts increased embryogenic callus formation on shoot apices and leaf explants while proline had no effect on embryogenic callus formation in medium with N6 salts. Increased growth with increasing proline concentration was obtained in suspension aggregates grown in medium with N6 salts, whereas proline only increased growth of suspension aggregates grown in medium with Murashige and Skoog salts at concentrations of 12.5 or 25 mM. A stimulating effect of proline on plant regeneration was observed in short-term cultures of callus as well as in long-term cultures of suspension aggregates. An optimum proline concentration for plant regeneration was found at 12.5 mM. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ammonium-induced proline and sucrose accumulation, and their significance in antioxidative activity and osmotic adjustment

Excess of ammonia generates oxidative and osmotic stress, and results in an accumulation of compatible solutes. The aim of this study was to investigate the physiological significance of excess ammonium-induced proline and sucrose accumulation on antioxidative activity and osmotic adjustment. The detached leaves of white clover (Trifolium repense L.) were fed with 0, 10, 50, 100, and 200 mM NH₄Cl, and the contribution of proline and sucrose to osmotic adjustment and their relationship with antioxidative enzymes activity were assessed. A gradual decline of relative water content and osmotic potential (Ψ_π) with increasing NH₄Cl feeding level was accompanied by an increase in ammonia concentration. Significant accumulation of proline and sucrose was observed when NH₄Cl was fed over 100 mM compared with control (0 mM NH₄Cl). The increase in enzyme activity was significant only at 200 mM for ascorbate peroxidase (APOD) and over 100 mM NH₄Cl for guaiacol peroxidase (GPOD) and catalase (CAT). The contribution of proline and sucrose to osmotic adjustment over 100 mM, where proline and sucrose accumulation was more important, maintained at control levels or significantly decreased. The content of proline and sucrose as affected by NH₄Cl feeding level was positively related with the activity of APOD, GPOD, and CAT. These results suggest that proline and sucrose accumulation induced by the excess of ammonium has a more influential role in antioxidative activity rather than osmotic adjustment.     

Glutamine synthetase and glutamate dehydrogenase contribute differentially to proline accumulation in leaves of wheat (Triticum aestivum) seedlings exposed to different salinity

To investigate the roles of ammonium-assimilating enzymes in proline synthesis under salinity stress, the activities of glutamine synthetase (GS; EC 6.3.1.2) and NADH-dependent glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) were determined in leaves of wheat (Triticum aestivum) seedlings exposed to salt stress at 150 and 300 mM NaCl for 5d. At the lower salinity, only GS activity increased markedly. At 300 mM NaCl, however, NADH-GDH activity increased while GS activity decreased. A significant accumulation of proline was found only at high-salinity exposure while glutamate, a proline precursor, increased dramatically under both low and high salinity. These data suggests that GS-catalysis might be the main glutamate synthesis pathway under low salinity. At 300 mM NaCl, glutamate seems to be preferentially produced through the process catalyzed by NADH-GDH. The increase of ammonium in salinity-stressed wheat seedlings might have resulted from increased photorespiration, which is responsible for the higher NADH-GDH activity. The activity of Delta(1)-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) was significantly enhanced at 300 mM NaCl but remained unchanged at 150 mM. Delta(1)-Pyrroline-5-carboxylate synthetase (P5CS) activity did not show a specific response, indicating that P5CR might be the limiting step in proline synthesis from glutamate at high salinity.     

Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of gamma-glutamyl kinase.

A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.     

Proline accumulation induced by weak acids and IAA in coleoptiles of wheat seedlings

Proline accumulation in coleoptiles of wheat seedlings or in excised coleoptile segments incubated under shaking for a 24 h period was studied. There was no increase of proline content of coleoptiles after incubation of the seedlings in 5 mM citric acid (a relatively strong and slowly penetrating organic acid) in a pH range from 4.5 to 7.0 and only a slight increase of proline content after incubation in phosphate buffer at pH 7.0 to 7.5 duo to the higher osmotic concentration of phosphate buffer in this pH range. Quite different results were obtained with seedlings incubated in 10 mM acetic acid, a weak and easily penetrating organic acid. With increasing proton concentrations, proline accumulation increased. Application of 400 mM mannitol or higher concentrations of IAA (more than 10⁻⁵M) additionally increased proline accumulation in the presence of 10 mM acetic acid in the pH range from 6.0 to 7.5 in which acetic acid alone was loss effective. It is suggested that a decrease of cytosolic pH causes stress—induced proline accumulation.     

Mechanisms of salt stress tolerance development in barley plants under the influence of 5-aminolevulinic acid

Growing barley (Hordeum vulgare L.) plants for 7 days on NaCl solutions (20–200 mM) decreased chlorophyll (Chl) a and b content with respect to that in untreated control plants. The content of free proline and the plant ability to synthesize 5-aminolevulinic acid (ALA) started to increase in parallel at salt concentrations of 20–50 mM. The maximum amount of ALA accumulated in plants grown at 100 mM NaCl was twofold higher than in control plants grown on fresh water. In this case the proline content increased 2.8-fold. On further increase in salt concentration, the rate of ALA accumulation decreased, approaching control values at 150 mM NaCl; even lower rates were observed at 200 mM NaCl. The reduced ability to synthesize ALA was accompanied by an increase in proline content. The albino tissue of plants treated at the seed stage with the antibiotic streptomycin lost its ability to synthesize ALA needed for Chl formation. The proline content in the albino tissue was tenfold higher than in control green plants and was 30-fold higher when the plants were grown on solutions with 100 mM NaCl. No effect of NaCl on ALA-dehydratase activity was noted. As NaCl concentration was raised, there occurred the decrease in magnesium chelatase activity, accumulation of reactive oxygen species (ROS), the increase in ascorbate peroxidase activity, and a slight decrease in lipid peroxidation level. Growing plants in the presence of 150 mM NaCl and 10 or 60 mg/l exogenous ALA led to the increase in proline content (by a factor of 1.8 and 4.2, respectively) and to the decrease in ROS content, in comparison with plants grown on salt solutions without ALA. Furthermore, in the presence of exogenous ALA, the parameters of seedling growth became similar to those of NaCl-untreated plants. The role of ALA in plants as an antistress agent is considered. ALA is supposed to confer tolerance to salt stress by taking part in Chl and heme biosynthesis and also through functioning as a plant growth regulator. A hypothesis is put forward that the impairment of ALA-synthesizing ability may redirect metabolic conversions of glutamic acid from Chl and heme synthesis to the proline synthesis pathway, which would stimulate proline biosynthesis and improve salt tolerance.     

Contribution of putrescine degradation to proline accumulation in soybean leaves under salinity

Proline accumulation was studied in the leaves of Glycine max (L.) Merr. subjected to salt stress in the presence of aminoguanidine (AG, a specific inhibitor of diamine oxidase, DAO) and exogenous putrescine (Put). Both DAO activity and proline content were increased while endogenous Put content was decreased in soybean leaves under 50 to 150 mM NaCl. There was a negative correlation between proline accumulation and endogenous Put content. The addition of AG during NaCl stress inhibited DAO activity, caused Put accumulation and a 15 to 20 % decrease in proline content. Application of 1 mM Put to NaCl solution markedly increased proline content. The promotive effect of Put application could be alleviated by the treatment with Put plus AG. Moreover an application of AG had no effect on proline accumulation in soybean seedlings grown under normal condition. These results indicate that the quantitative contribution of Put degradation to proline formation is 15 to 20 %.     

Proline antioxidant role in the common ice plant subjected to salinity and paraquat treatment inducing oxidative stress

Leaves of 4-week-old (juvenile) and 9-week-old (adult) plants of the halophyte Mesembryanthemum crystallinum L. (the common ice plant), cultured under controlled conditions in the phytotron, were treated with paraquat (0.1 μM), which produces superoxide radical, and (or) paraquat combined with introduction of NaCl (100 mM) or proline (5 mM) into nutrient medium. After a 20-h dark period (23°C), plants were transferred into light (4 h at 54.1 W/m² of photosynthetically active radiation) for stimulation of O°₂⁻ formation in plastids. Activities of antioxidant enzymes, the contents of MDA, H₂O₂, chlorophyll, and free proline were measured in leaves. Plant responses in two age groups, which differed in the type of photosynthesis (juvenile plants had C₃ type of photosynthesis, whereas adult plants were at the transition stage to Crassulacean Acid Metabolism (CAM) photosynthesis), differed in the levels of constitutive proline and proline, induced by NaCl and paraquat, as well as in activities of superoxide dismutase (SOD) and catalase. Changes in SOD activity and proline accumulation in response to paraquat treatment combined with NaCl revealed opposite dependence to accumulation of proline: the more proline accumulated in leaves, the lower activity of the enzyme. In response to paraquat treatment, the content of chlorophylls a and b most drastically declined in juvenile plants. Negative effect of salinity on the content of chlorophylls was lower than that of paraquat and was almost the same in plants of both age groups. Protective effect of exogenous proline was most profound in the case of paraquat treatment. Exogenous proline decreased the rate of lipid peroxidation, the content of superoxide radical and, consequently, SOD activity (almost fivefold), and increased the content of chlorophylls (a and b) in leaves of adult plants. The obtained data suggest that stress-induced accumulation of proline in the common ice plant has both osmoprotectory and antioxidant functions.     

Proline biosynthesis in a proline-accumulating barley mutant

A barley (Hordeum vulgare L.) mutant, R5201, selected for resistance to 4? mM trans-4-hydroxyproline had a 3–6 fold increase in the soluble proline content of the leaf compared with the parent cultivar, Maris Mink. The mutant converted more [U-⁴C]glutamic acid to free proline in the leaves than Maris Mink but incorporation into protein proline was similar. Incorporation of radioactivity into proline was inhibited by exogenous proline more in Maris Mink than R5201, suggesting that feedback inhibition of proline biosynthesis is relaxed, but not absent in the mutant. When [1-¹⁴C]ornithine was the precursor, both R5201 and Maris Mink incorporated similar small amounts of label into soluble and protein proline. More protein proline was formed by both genotypes from labelled glutamic acid than from labelled ornithine. There may exist two routes of proline formation, where the glutamate pathway is synthetic and the ornithine pathway is catabolic.     

Effect of chemical chaperone addition on production and aggregation of recombinant flag-tagged COMP-angiopoietin 1 in Chinese hamster ovary cells

To investigate the effect of chemical chaperones on the production and aggregation of flag-tagged cartilage oligomeric matrix protein-Angiopoietin1 (FCA1) in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in serum-free media with various chemical chaperones, 1 mM 4-phenylbutyrate (4-PBA), 200 mM proline, 3% glycerol, 2% dimethyl sulfoxide (DMSO), and without chemical chaperone as control. The addition of chemical chaperones enhanced FCA1 production and specific FCA1 productivity, q(FCA)(1). For example, the q(FCA)(1) at 200 mM proline was fourfold higher than that at control. Unlike q(FCA)(1), the aggregation of FCA1 was strongly affected by which chemical chaperone was added. The addition of 2% DMSO and 200 mM proline significantly reduced the proportion of aggregates, but the addition of 1 mM 4-PBA and 3% glycerol was hardly effective. The proportions of aggregates were 29.5 and 55.6% at 2% DMSO and 200 mM proline, respectively, whereas it was 79.6% at control. The exact mechanism how chemical chaperones affected the aggregation of FCA1 was not investigated in this study, and therefore, more extensive works will be needed to clarify why different chemical chaperones behaved differently in reducing the aggregation of FCA1. Among chemical chaperones tested, DMSO was the most effective one in regard to enhancing the production and reducing the aggregation of FCA1 in CHO cells.     

Effect of amino acids on cryopreservation of cynomolgus monkey (Macaca fascicularis) sperm

The effects of three amino acids (proline, glutamine, and glycine) added to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa were studied. This is the first report on the effects of amino acids on nonhuman primate sperm cryopreservation. The addition of 5mM proline, 10mM glutamine, and 10 or 20mM glycine each significantly improved post-thaw sperm motility and membrane and acrosome integrity compared with the control (TTE alone). However, a significant decrease in motility and membrane/acrosome integrity was observed when amino acid concentrations increased to 60mM for proline and glutamine, and 80 mM for glycine. The results suggest that adding a limited amount of amino acids to the freezing media is beneficial for freezing cynomolgus monkey sperm.