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PubMed1995

The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12.

The ProP and ProU transport systems of Escherichia coli mediate the uptake of several osmoprotectants including glycine betaine. Here we report that both ProP and ProU are involved in the transport of the potent osmoprotectant proline betaine. A set of isogenic E. coli strains carrying deletions in either the proP or proU loci was constructed. The growth properties of these mutants in high osmolarity minimal media containing 1 mM proline betaine demonstrated that the osmoprotective effect of this compound was dependent on either an intact ProP or ProU uptake system. Proline betaine competes with glycine betaine for binding to the proU-encoded periplasmic substrate binding protein (ProX) and we estimate a KD of 5.2 microM for proline betaine binding. This value is similar to the binding constant of the ProX protein determined previously for the binding of glycine betaine (KD of 1.4 microM). Our results thus demonstrate that the binding-protein-dependent ProU transport system of E. coli mediates the efficient uptake of the osmoprotectants glycine betaine and proline betaine.

Amino Acid Transport Systems↗
PubMed1988

The Escherichia coli proB gene corrects the proline auxotrophy of Saccharomyces cerevisiae pro1 mutants.

We constructed plasmids carrying the Escherichia coli proB gene that encodes gamma-glutamyl kinase, under the control of the yeast GAL1 promoter. This construction was carried out with both the wild-type proB+ gene and a mutant allele, proB74, that specifies an enzyme resistant to feedback inhibition by proline. Yeast pro1 mutants harboring these plasmids are proline prototrophs. We conclude that the pro1 mutation results in a deficiency in the gamma-glutamyl kinase activity in Saccharomyces cerevisiae. Expression of the proB74 allele in yeast resulted in enhanced resistance to the proline analogue L-azetidine-2-carboxylate and in a 2.4-fold elevation of the intracellular free proline levels. This result suggests that gamma-glutamyl kinase is the rate limiting step in proline biosynthesis in yeast.

Alleles↗
PubMed1995

Proline betaine is a highly effective osmoprotectant for Staphylococcus aureus.

Proline betaine is an osmoprotectant that is at least as effective as glycine betaine, and more effective than L-proline, for various strains of Staphylococcus aureus, and Staphylococcus epidermidis and Staphylococcus saprophyticus. 13C NMR studies revealed that proline betaine accumulated to high levels in osmotically stressed S. aureus, but was also detected in organisms grown in its presence in the absence of osmotic stress. Competition experiments indicated that proline betaine was taken up by the proline transport systems of S. aureus, but not by the high affinity glycine betaine transport system.

Betaine↗
PubMed1981

Proline over-production results in enhanced osmotolerance in Salmonella typhimurium.

Mutants of S. typhimurium with enhanced osmotolerance were isolated. These mutants were obtained as strains which over-produced proline due to regulatory mutations affecting proline biosynthesis. The mutations are located on F'proBa and upon transfer to other S. typhimurium strains, they confer enhanced osmotolerance on the recipients. The osmotolerant mutants not only have higher intracellular proline levels than the osmosensitive parental strain, but the proline levels in the osmotolerant mutants are regulated such that they increase in response to osmotic stress. Possibly reasons why elevated proline levels lead to enhanced osmotolerance are discussed.

Amino Acids↗
PubMed1976

Mitochondrial proline dehydrogenase deficiency in hyperprolinemic PRO/Re mice: genetic and enzymatic analyses.

Genetic analyses, involving backcross and F2 matings, demonstrate that the type I hyperprolinemia of PRO/Re mice is caused by an abnormal allelet at a single locus designated pro-1. Mice homozygous for this allele (pro-1b/pro-1b) posses a deficiency in the activity of component 1 of mitochondrial proline dehydrogenase. In liver mitochondria of normal C57BL/6J mice, two proline dehydrogenase activity components are demonstrable by electrophoretic resolution of Triton X-100 solubilized extracts. In mitochondria of PRO/Re mice, the activity of component 1 is not readily detectable. Residual proline dehydrogenase activity in PRO/Re mitochondria appears, therefore, to be due in large measure to activity component 2 which is more stable to incubation at 40 C, exhibits slower electrophoretic mobility, and is less reactive to menadione. Kinetic analyses demonstrate a Km (proline) for the Triton X-100 solubilized enzyme activities of PRO/Re and C57BL/6J liver mitochondria of 0.4 M and 2.9 X 10(-3) M, respectively. C57BL/6J enzyme activity is inhibited by high substrate concentration. The actins of PRO/Re liver obtained by differential centrifugation. Abnormal control of respiratory chain function in PRO/Re mitochondria appears to involve primarily proline oxidation, as indicated by the level of activity of several inner membrane enzymes.

Alleles↗
PubMed1979

Kinetics of L-proline reabsorption in rat kidney studied by continuous microperfusion.

Renal tubular reabsorption of 3H and 14C labelled L-proline was measured in vivo et situ by continuous microperfusion of single proximal tubules of the rat. The reabsorption is shown to be saturable. Passive diffusion plays a relatively small role in the reabsorption. A maximum possible permeability coefficient of 25 micrometers 2.s-1 for proline was calculated. Two transport systems were found, one with a small affinity and a high capacity, the other with a very high affinity and a small capacity. The following values were estimated. Jmax 1 = 2.6 +/- 0.28 (SEM) nmol.m-1.S-1 Km1 = 11.8 +/- 1.7 (SEM) mmol.1-1 Jmax 2 = 9.6 +/- 1.92 (SEM) pmol.m-1.s-1 Km2 = 29.3 +/- 7.8 (SEM) mumol.1-1. Whereas the first system reabsorbs the bulk of the filtered load, the activity of the second system explains the extremely small amount of proline found in the final urine. Diisopropylphosphorofluoridate--a specific inhibitor of dipeptidyl peptidase IV--decreases the reabsorption of L-proline and L-alanine but has no influence on the reabsorption of the basic amino acid L-arginine and the acidic amino acid L-glutamic acid. This result correlates with a recent speculation that dipeptidyl peptidase IV is involved in proline and alanine reabosrption.

Animals↗
PubMed1989

High-affinity binding of proline to mouse brain synaptic membranes.

There is evidence suggestive of the possible neuromodulatory role for L-proline in the mammalian brain. The binding of proline to whole mouse brain synaptic membranes has been partially characterized. Several binding sites for this imino acid have been identified; one in the nanomolar range and at least two in the submicromolar range. The binding of proline is inhibited by NaCl. Pipecolic acid (40 microM), ornithine, aminooxyacetic acid (AOAA), glycine, GABA, and glutamate were capable of significantly inhibiting proline binding. Although detailed pharmacological and functional studies are needed, these results are consistent with a brain-specific function for this imino acid, as well as, with the presence of specific binding site(s) for proline.

Amino Acid Transport Systems, Neutral↗
PubMed1977

Variant Chinese hamster cells resistant to the proline analog L-azetidine 2-carboxylic acid.

Variants resistant to the toxic effects of the proline analog L-azetidine 2-carboxylic acid (AZCA) have been isolated from the Chinese hamster tissue culture line G3 by a three-step selection procedure using increasing concentrations of AZCA. Cells surviving each of the three selective steps have been examined for AZCA resistance and for proline uptake, biosynthesis, and degradation. The largest increment in AZCA resistance is acquired in the third step and is due to overproduction of proline as a result of increased activity of the enzyme system responsible for the conversion of glutamic acid to glutamic gamma-semialdehyde. It is not accompanied by an increase in the rate of formation of proline from ornithine or in the rate of proline uptake or degradation.

Azetidinecarboxylic Acid↗
PubMed1991

Kinetic analysis of experimental post-operative peritoneal healing: the incorporation of proline and glucosamine by exudative and tissue repair cells.

The purpose of this study was to experimentally investigate the cellular composition of post-surgical peritoneal fluid and peritoneal tissue and determine the patterns of 14C-proline and 14C-glucosamine incorporation by the peritoneal exudative cells and peritoneal tissue repair cells (PEC and PTRC). One group of rabbits underwent resection (2.0 cm) and reanastomosis of their ileum, and another group underwent peritoneal wall abrasion. Postoperatively (1-28 days), the PEC and PTRC were collected and incubated for 5 days with 0.5 mu Ci 14C-glucosamine or 14C-proline, and the specific activity thereafter determined by beta counting. On the 1st postoperative day, the total cell number (TCN) has increased (7.7 x 10(7) cells/rabbit) to 770 per cent of the control values primarily as a result of the PMN influx (89.9 per cent). On day 3, the TCN was 6.1 x 10(7), 58.5 per cent of which comprised macrophages, which had become the principle cell type by day 5. The incorporation of proline and glucosamine into the PEC increased significantly peaking on day 7 then decreasing to the control value by day 21. Proline incorporation into the PTRC increased significantly, reaching a peak value on day 5, which decreased by day 10. Glucosamine incorporation reached a peak value on day 7 then decreased by day 10. In conclusion, the increase in glucosamine and proline incorporation into the PTRC parallels the increase in PEC, comprised principally of macrophages. These findings suggest that analysis of the metabolic activities in peritoneal activated macrophages may provide a useful tool to dissect the central mediation of postsurgical peritoneal re-epithelialization.

Animals↗
PubMed1993

Improvement of culture conditions for L-proline production by a recombinant strain of Serratia marcescens.

Serratia marcescens SP511 was previously reported to be an L-proline-producing strain that harbors a recombinant plasmid carrying the mutant type of the proline operon. This strain produced 65 g/L of L-proline in a medium containing 22% sucrose and urea after 5 d of incubation under the conventional culture conditions. We searched for more suitable culture conditions for more abundant L-proline production by SP511. To improve the supply of a nitrogen source to cells, ammonium was used instead of urea and fed to a culture under control of the pH of the medium. The concentrations of MgSO4 and K2HPO4 were increased, and in addition, sucrose was continuously added to the culture at a final concentration of 32%. Under these conditions, the cell amount was increased twofold over that under the previous conditions and L-proline production reached a maximum of more than 100 g/L after 4 d of incubation.

Ammonia↗
PubMed2000

Distributions of p53 codon 72 polymorphism in bladder cancer--proline form is prominent in invasive tumor.

Abnormal function of p53 is commonly associated with various cancer formations. High-grade and late-stage bladder cancers have been reported to have mutated or become inactive p53 when using immunohistochemical stains. Recently, p53 codon 72 polymorphism was extensively studied to determine the risk factors responsible for cancer formation. There was a general population of codon 72 sequence polymorphism of the wild type p53. A single base change from G to C caused the alteration of amino acid residue 72 from arginine to proline. The arginine form is considered to be a significant risk factor in the development of cancer. However, various reports had indicated discrepancies with regard to this polymorphism; some showed no significant difference between the control and cancer groups, while other series were associated with high risks in the proline form homozygotes. To resolve the undefined distribution of this polymorphism in bladder cancers, 58 patients with bladder cancer were enrolled onto this study. When checked using the Chi-squared test (P = 0.952) there were no differences between the control subjects and bladder cancer patients in the distribution of polymorphism. However, proline form homozygotes were more frequently found in the invasive group than the non-invasive group by Fisher's exact test (25% and 2.9%, respectively, P < 0.001). More than 70% of the non-invasive bladder cancers were the arginine form homozygotes. This result is consistent with those reported for hepatocellular carcinoma that showed a history of chronic liver disease and proline form homozygotes in a report by Yu et al. Our data suggest that proline form homozygotes are associated with invasive bladder cancer.

Adult↗
PubMed2003

Gene and primary structures of dye-linked L-proline dehydrogenase from the hyperthermophilic archaeon Thermococcus profundus show the presence of a novel heterotetrameric amino acid dehydrogenase complex.

Dye-linked l-proline dehydrogenase catalyzes the oxidation of l-proline in the presence of artificial electron acceptors such as 2, 6-dichloroindophenol and ferricyanide. The enzyme from the hyperthermophilic archaeon Thermococcus profundus was purified and characterized for the first time in archaea by Sakuraba et al. in 2001. In this study, cloning and sequencing analyses of the gene encoding the enzyme and functional analysis of the subunits were performed. The gene formed an operon that consisted of four genes, pdhA, pdhB, pdhF, and pdhX, which are tandemly arranged in the order of pdhA-F-X-B. SDS-PAGE analysis of the purified recombinant enzyme showed four different bands corresponding to alpha (54 kDa), beta (43 kDa), gamma (19 kDa), and delta (8 kDa) subunits encoded by pdhA, pdhB, pdhF, and pdhX, respectively, and the molecular ratio of these subunits was determined to be equal. This indicates that the enzyme consists of a heterotetrameric alphabetagammadelta structure. Functional analysis of each subunit revealed that the beta subunit catalyzed the dye-linked l-proline dehydrogenase reaction by itself and that, unexpectedly, the alpha subunit exhibited dye-linked NADH dehydrogenase activity. This is the first example showing the existence of a bifunctional dye-linked l-proline/NADH dehydrogenase complex. On the basis of genome analysis, similar gene clusters were observed in the genomes of Pyrococcus horikoshii, Pyrococcus abyssi, Pyrococcus furiosus, and Archaeoglobus fulgidus. These results indicate that the dye-linked l-proline dehydrogenase is a novel type of heterotetrameric amino acid dehydrogenase that might be widely distributed in the hyperthermophilic archaeal strain.

Amino Acid Sequence↗
PubMed2004

Influence of proline on rat brain activities of alanine aminotransferase, aspartate aminotransferase and acid phosphatase.

Hyperprolinemia type II (HPII) is an autosomal recessive disorder caused by the severe deficiency of enzyme delta1-pyrroline-5-carboxylic acid dehydrogenase leading to tissue accumulation of proline. Chronic administration of Pro led to significant reduction of cytosolic ALT activity of olfactory lobes (50.57%), cerebrum (40%) and medulla oblongata (13.71%) only. Whereas mitochondrial ALT activity was reduced significantly in, all brain regions such as olfactory lobes (73.23%), cerebrum (70.26%), cerebellum (65.39%) and medulla oblongata (65.18%). The effect of chronic Pro administration on cytosolic AST activity was also determined. The cytosolic AST activity from olfactory lobes, cerebrum and medulla oblongata reduced by 75.71, 67.53 and 76.13%, respectively while cytosolic AST activity from cerebellum increased by 28.05%. The mitochondrial AST activity lowered in olfactory lobes (by 72.45%), cerebrum (by 78%), cerebellum (by 49.56%) and medulla oblongata (by 69.30%). In vitro studies also showed increase in brain tissue proline and decrease in glutamate levels. In vitro studies indicated that proline has direct inhibitory effect on these enzymes and glutamate levels in brain tissue showed positive correlation with AST and ALT activities. Acid phosphatase (ACP) activity reduced significantly in olfactory lobes (40.33%) and cerebrum (20.82%) whereas it elevated in cerebellum (97.32%) and medulla oblongata (76.33%). The histological studies showed degenerative changes in brain. Following proline treatment, the animals became sluggish and showed low responses to tail pricks and lifting by tails and showed impaired balancing. These observations indicate influence of proline on AST, ALT and ACP activities of different brain regions leading to lesser synthesis of glutamate thereby causing neurological dysfunctions.

Acid Phosphatase↗
PubMed1983

Demonstration of proline-rich proteins in rabbit parotid saliva and partial characterization of some of the proteins.

Rabbit parotid saliva was collected by cannulation of the secretory duct in anaesthetized animals. Proteins which cross-react with antibodies to human acidic proline-rich proteins were demonstrated in the secretion and fractionated by chromatography on an immunosorbent and CM32-cellulose. At least 5 proline-rich proteins were identified with molecular weights ranging from 19,000 to 61,000 as determined by sodium dodecylsulphate acrylamide gel electrophoresis. The major immunoreactive components were basic proteins with similar size and charge properties. In two of the proteins, proline, glycine and glutamic acid or glutamine accounted for 84 or 99 per cent of all residues. In contrast to proline-rich proteins from other species, proline constituted only 13 or 17 per cent of total amino acids.

Amino Acids↗
PubMed1978

Characteristics of proline transport in normal and starved cells of Candida albicans.

1. There was no apparent correlation between the rate of respiration and rate of accumulation of proline in Candida albicans cells. 2. In contrast to normal cells, the respiration in the starved cells became completely cyanide insensitive. The starvation of cells in the presence of cycloheximide prevented the cells from becoming cyanide insensitive. The addition of Fe(III), however, accelerated the process. 3. Oxidizable substrates e.g. NADH, acetate and glucose, when added to cyanide-insensitive starved cells, exhibited 40--280% stimulation in respiration rate. However, this enhancement in oxidation by various substrates was not coupled to a simultaneous increase in the proline uptake or in intracellular ATP levels. 4. There was 6-fold stimulation in proline uptake when cyanide-insensitive cells were preincubated with 50 mM glucose. The preincubation of starved cells resulted in a partial restoration of cyanide sensitivity and increased intracellular ATP levels. The preincubation of starved cells with other oxidizable substrates resulted in a partial restoration of cyanide sensitivity but had no stimulatory effect on intracellular ATP levels and proline accumulation. 5. Both the enhanced uptake and ATP levels in glucose preincubated cells were found to be completely abolished by iodoacetate. 6. It is proposed that the increased proline uptake in cells preincubated with glucose was mainly due to the production of glycolytic energy.

Adenosine Triphosphate↗
PubMed1992

Proline uptake by monolayers of human intestinal absorptive (Caco-2) cells in vitro.

Monolayers of the Caco-2 human intestinal cell line exhibit active and passive uptake systems for the imino acid L-proline. The active transport component is saturable and it is responsible for about two thirds of the observed flux over the nanomolar concentration range, at 37 degrees C and pH 7.4. In contrast to L-phenylalanine, specific L-proline uptake has a high degree of sodium dependency and the efficiency of the carrier system is significantly reduced when protein synthesis (cycloheximide), Na+/K(+)-ATPase (ouabain) or cellular metabolism (sodium azide) are inhibited. The expression of the L-proline carrier by Caco-2 cells was under some degree of nutritional control. Glucose deficiency, over the time scale of the experiment, had no effect. The temperature-dependence of the specific uptake process followed the Arrhenius model with an apparent activation energy of 93.5 kJ nmol-1. This pathway also displayed Michaelis-Menten concentration-dependence with a Ksdm of 5.28 mM and a maximal transport flux (Jsdmax) of 835 pmol min-1 (10(6) cells)-1. Although the passive component was unchanged, the pH of the donor phase exerted a profound effect on the active carrier component. Within the physiological pH range a local maximum efficiency was found at pH 7.4 but dramatic increases were noted as pH 5.0 was approached. In competition studies, with 100-fold excess of a second amino acid, strong inhibition of uptake was found with alpha-aminoisobutyric acid, L-alanine and L-serine whereas moderate inhibition was observed with glycine, D-proline and gamma-aminoisobutyric acid. Aromatic and branched amino acids showed weak (L-valine) or no interaction (L-phenylalanine, L-leucine) with the carrier system. These data indicate that the carrier system for the uptake of L-proline has many features in common with the A system for amino acid transport.

Azides↗
PubMed1979

Action of thiazolidine-2-carboxylic acid, a proline analog, on protein synthesizing systems.

Thiazolidine-2-carboxylic acid, or beta-thiaproline, is a proline analog in which the beta methylene group of proline is substituted by a sulfur atom. It has been deomonstrated that beta-thiaproline is activated and transferred to tRNAPro by Escherichia coli and rat liver aminoacyl-tRNA synthetases, and inhibits proline incorporation into polypeptides in protein synthesizing systems from E. coli, rat liver or rabbit reticulocytes. In mammalian systems beta-thiaproline inhibits also leucine incorporation; in rabbit reticulocyte lysate it inhibits ribosome run-off. Both these effects may be explained by the fact that beta-thiaproline once incorporated into the growing polypeptide chain impairs its further elongation, as shown by experiments made with puromycin. All tests were performed in comparison with thiazolidine-4-carboxylic acid, or gamma-thiaproline, another proline analog having the gamma methylene group substituted by a sulfur atom; it was shown that in all the reactions studied both compounds act as competitive inhibitors of proline. Some differences in the effects of the two analogs have been evidenced: in almost all the reactions and mainly in the whole protein synthesizing systems, beta-thiaproline shows an higher inhibitory activity.

Amino Acids, Sulfur↗
PubMed1976

Transport of L-proline by rat brain slices.

L-Proline is taken up into slices of rat cerebral cortex by a structurally specific 'high affinity' system which is absolutely dependent on sodium ions. The system mediating L-proline uptake in homogenates of cerebral cortex is associated with osmotically sensitive particles of the same equilibrium density as synaptosomes. Based on tissue-medium ratios, the uptake of L-proline is most efficient in slices of cerebral cortex and of hypothalamus, and least efficient in slices of cerebellum. L-Proline taken up into slices of cerebral cortex can be released from these slices by an increased potassium ioort into and out of brain slices are consistent with L-proline functioning as a synaptic transmitter in that they are analogous to observations on the transport of transmitter amino acids such as GABA.

Amino Acids↗