Concentration Of L-proline Research Articles

Interleukin-1β-induced changes in the kinetic constants of l-proline uptake in human skin fibroblasts

The effects of interleukin-1 beta (IL-1) on L-proline uptake in human skin fibroblasts were investigated. Exposure of the fibroblasts to IL-1 (5, 10 or 50 pg/ml) for 2 h did not change L-proline uptake. In contrast, inhibition was observed after 6 h of IL-1 treatment, and only 60% of the control uptake remained after incubation for 24 h with 10 pg of IL-1/ml. IL-1 depressed the activity of both transfer systems; the low-affinity system inhibited by alpha-(methylamino)isobutyric acid (Me-AIB), corresponding to system A, and a high-affinity transfer system which is unaffected by Me-AIB. The inhibitory effect increased as the L-proline concentration decreased. To determine whether IL-1-induced prostaglandin release influences proline uptake, indomethacin (14 microM) was added as a cyclo-oxygenase inhibitor. Indomethacin itself decreased L-proline uptake but to a lesser extent than did IL-1. When IL-1 was tested in the presence of indomethacin, the inhibition of L-proline uptake was still observed, with values between those obtained with each substance in isolation. This suggests that the inhibitory effect of IL-1 on proline uptake by skin fibroblast does not only involve the prostaglandins that accumulate in the medium, but no firm conclusion can be drawn, due to the fact that the inhibition by the two agents was not statistically independent. Kinetic analyses for 1 min combined with inhibition experiments showed that IL-1 induced a decrease in the Km and Vmax, values of the high-affinity transport system, whereas it increased the Km of system A. Therefore the two systems of proline uptake in skin fibroblasts are probably inhibited by IL-1 via different mechanisms.

Analysis of the Serratia marcescens proBA operon and feedback control of proline biosynthesis

The proBA operon, coding for gamma-glutamyl kinase (GK) and gamma-glutamyl phosphate reductase (GPR), was cloned from the chromosome of the wild-type strain of Serratia marcescens Sr41. From our sequence data the proB (1101 bp) and proA (1472 bp) genes were shown to code for two proteins of Mr 39,169 and 44,640, respectively. Analysis of expression of the lacZ structural gene fused with the proBA promoter showed that the proBA operon is not subject to proline-mediated feedback repression. Amplification of the proBA operon enabled us to determine GK activity, which was inhibited in the presence of a low concentration of L-proline. Comparison of the amino acid sequences of the S. marcescens GK and GPR proteins with those of the GK and GPR proteins from E. coli revealed extensive similarities.

Schistosoma mansoni: Higher free proline levels in the livers of infected mice

The concentration of l-hydroxyproline in the liver of ICR female mice increased rapidly during the 8th to 11th weeks of Schistosoma mansoni infection. Free l-proline concentration began to increase about the 7th week and reached its maximum at the 8th to 9th weeks of the infection, when the granulomatous response to the schistosome eggs in the liver was most prominent, as indicated by the increase in liver wet weight and its deoxyribonucleic acid concentration. A significant increment in the total activity of ornithine-δ-transaminase (EC 2.6.1.13) and the decrease in the specific activity of proline oxidase (EC 1.4.3.2) became detectable in the liver homogenate of infected mice on the 8th week. However, changes in these enzymatic activities were not parallel to that of the hepatic free l-proline content. Intraperitoneal administration of S. mansoni egg granulomas or 15,000 g × 30 min supernatant fluid of their extracts into uninfected, normal mice significantly increased the hepatic free l-proline content without any appreciable effect on the enzymatic activities of proline oxidase and ornithine-δ-transaminase. These findings suggest that S. mansoni egg granulomas contain a factor(s) which may be responsible for the elevation of free l-proline content in the fibrotic liver caused by experimental schistosomiasis mansoni.

Stimulation by L-Proline of 5'-Inosinic Acid Production by Mutants of Corynebacterium ammoniagenes.

The effects of amino acids on IMP production were examined with a mutant strain, KY10895, derived from Corynebacterium ammoniagenes KY13374. l-Proline improved the productivity of IMP more than any other amino acid. The optimum concentration of l-proline for IMP production was 1–2% and the IMP productivity was about 70% more than that in the control medium. The effects of l-proline analogs on IMP production were also examined with the mutant KY10895. DL-3,4-Dehydroproline inhibited IMP production. Mutants resistant to growth inhibition by dl-3,4-dehydroproline were derived from strain KY10895. Among mutants thus obtained, strain H-7335 had the highest productivity. The intracellular concentrations of l-proline in strain H-7335 were higher than those of the parental strain, KY10895. These findings indicated that an increase in intracellular l-proline was linked with an increase of IMP productivity and strengthening the l-proline synthesis of a strain was an effective method for obtaining a hyper-producer o...

Inhibition Of Cell Division By Proline Analogues: Reversal by Proline and High Salinity

Summary Six proline analogues were tested for their ability to inhibit cell division in N. bacillaris at low salinity. Two analogues, thiazolidine-4-carboxylic acid (T4C) and azetidine-2-carboxylic acid (A2C) were found to be effective inhibitors. Inhibition by 1 mM T4C was not readily reversed by L-proline while inhibition by 20 mM A2C was substantially reversed by an equimolar concentration of L-proline. Also, inhibition by T4C was partially reversed by L-asparagine and Lglutamine and inhibition due to A2C by L-alanine and L-glutamine. When cells were grown in high salinity media, sensitivity to inhibition by T4C increased. Conversely, A2C became ineffective as an inhibitor of cells at high salinity. Inasmuch as N. bacillaris accummulates high intracellular concentrations of free proline when grown at high salinity, the reversal of inhibition by A2C at high salinity may have been due to the high intracellular pool of proline in such cells.

Identification of osmoresponsive genes in Escherichia coli: evidence for participation of potassium and proline transport systems in osmoregulation.

Mu d1(Ap lac)-generated operon fusions were used in the identification of genes in Escherichia coli whose transcriptional expression is altered by changes in the osmolarity of the growth medium. One such osmoresponsive gene, designated osrA, was induced 400-fold when the osmolarity of the medium was increased with the addition of either ionic or neutral impermeable solutes but was not induced with glycerol, which is freely permeable across the cell membrane. osrA was mapped to 57.5 min and was shown to be transcribed clockwise on the E. coli chromosome. The ability of small concentrations of L-proline to promote the growth of E. coli in high-osmolar medium was shown to have been specifically lost in osrA mutants; other lines of evidence were also obtained to support the notion that osrA codes for an osmoresponsive L-proline transport system and is homologus to proU in Salmonella typhimurium. A second osmoresponsive operon identified was kdp, which codes for an inducible K+-transport system in E. coli. kdp expression was elevated 12-fold when the osmolarity of the growth medium was increased with the addition of impermeable ionic solutes but not neutral solutes; furthermore, osmoresponsivity of kdp expression was demonstrable only in K+-limiting media. kdp mutants were able to grow normally in high-osmolar media, but strains defective in both kdp and trkA (a gene for a second major K+-transport system) displayed an osmosensitive phenotype. The results suggest that transport systems for L-proline and K+, specified by osrA (proU) and kdp, respectively, play independent and important roles in osmoregulation in E. coli. A third osmoresponsive gene that was identified was lamB, which codes for an outer membrane protein for maltodextrin transport and lambda phage adsorption; its expression was reduced fourfold with increase in the osmolarity of the growth medium.

Physical mechanism for regulation of proton solute symport in Escherichia coli.

The activity of the Escherichia coli transport proteins for lactose and proline can be altered by changing the redox state of the dithiols in these carriers. A series of lipophilic oxidizing agents has been shown to inhibit and subsequent addition of dithiothreitol to restore full activity. Both systems are irreversibly inhibited by N-ethylmaleimide, but prior addition of oxidizing agents protects against this inhibition. These data, as well as studies on the inhibitory effect of the dithiol-specific reagent phenylarsine oxide, show that the redox-sensitive step is the conversion of a dithiol to a disulfide. Measurement of the initial rate as a function of the lactose and L-proline concentrations shows that the oxidation of a dithiol to a disulfide increases the Km of the carriers for lactose and L-proline. The reduced (dithiol) form of the carrier has a low Km and the oxidized (disulfide) form has a high Km for its substrate. The changes in Km brought about by reduction and oxidation are the same as those that accompany the generation and dissipation, respectively, of an electrochemical proton gradient (delta mu H+). These results support a mechanism in which an delta mu H+ or one of its components alters the ligand affinities of the carrier during a single transport cycle through conversion from the reduced to the oxidized form.

Glutamate agonistic and antagonistic activity of l-proline investigated in the hippocampal slice

l-Proline in concentrations of 5 mM and more acted as a glutamate antagonist, reversibly blocking transmission in synapses between the Schaffer collaterals and pyramidal dendritic spines in CA1 of the rat hippocampal slice, probably by competing with glutamate for glutamate receptors in the postsynaptic membrane. This explanation was supported by the partial reversal of the proline-induced synaptic blockade by 4-aminopyridine, which enhances spike duration and presumably increases the release of the natural transmitter. Lower l-proline concentrations (1–3 m m) promoted evoked population spike activity. This was postulated to be due to a glutamate agonistic effect of l-proline which, bound to glutamate receptors, would cause depolarization of dendritic plasma membranes and K + release. The effect of l-proline would be similar to that of glutamate, but less effective. An increase of the K + concentration in the superfusion solution, which also can be expected to cause depolarization, had a similar enhancing effect on the population spike activity. A reduction of the K + concentration depressed this activity. The possibility was considered that the promotion of population spikes by 1–3 m ml-proline is due to the suppression of the effect of inhibitory interneurons (basket cells) on the CA1 neurons. However, abolishing this inhibition by 10 −4 m bicuculline did not prevent the facilitating effect of l-proline on the population spikes, which seems to exclude this explanation.

Proline uptake in Candida albicans.

L-Proline entered both mycelial and yeast cells of Candida albicans by an active transport system of high specificity at low (less than 0.1 mM) external concentrations of substrate. The apparent Km value of this system was 0.1 mM for both types of cells, while the V value was 4 nmol min-1 (mg dry wt)-1 for mycelial cells and 1.4 nmol min-1 (mg dry wt)-1 for yeast cells. At L-proline concentrations greater than 0.1 mM, the amino acid appeared to enter both morphological forms by diffusion as well as active transport. As saturation was approached diffusion became increasingly important. The higher uptake rate of mycelial cells seemed not to be the result of an inducible system. The optimal pH and temperature for transport of L-proline were 7.0 and 37 degrees C, respectively. Sodium azide and the proline analogues sarcosine and L-azetidine-2-carboxylic acid inhibited L-proline uptake, while L-thiazolidine-4-carboxylic acid was less effective. The active transport system was highly specific for L-proline since neither ammonium ions, which inhibit the general amino acid transport system of fungi, nor 16 different amino acids interfered substantially with uptake.

L-proline transport by brush border membrane vesicles prepared from human placenta.

1. Brush border microvillous plasma membranes were prepared from syncytiotrophoblast of human term placenta by a method of differential centrifugation. 2. Such plasma membranes form closed, osmotically active, right-side-out vesicles into which L-proline (188 microM) is shown to be transported in a time-dependent manner. There is no detectable metabolism of L-proline within the vesicles during 30 min of incubation. 3. Transient accumulation of L-proline to levels of up to three times its equilibrium value occurs in the presence of an inward gradient of sodium chloride. The proline and sodium are shown to have reached electrochemical equilibrium by 30 min, at which stage there is about 100 pmol L-proline mg protein-1. 4. This transient accumulation is abolished by the prior equilibration of the sodium chloride gradient, or by the replacement of sodium by inwardly directed gradients of other cations. Entry of the amino acid into the vesicles is also shown to be influenced by the permeability of the anion in the medium and by an imposed potassium diffusion potential. L-Proline transport across the brush border membrane of human placental syncytiotrophoblast is thus a sodium-dependent, electrogenic process. 5. Studies of the transport processes indicate saturation at higher concentrations of L-proline with a 'Km' of about 1 mM; Vmax averaged about 2 nmol mg protein-1 min-1 varied considerably between preparations. 6. L-Proline (188 microM) transport is inhibited competitively by the presence of many amino acids and by the dipeptide glycyl-L-proline. The Ki for inhibition by methyl AIB is 300 microM. 7. These findings are discussed in relation to the mechanism of transplacental amino acid transfer.

Na +-gradient-dependent transport of l-proline and analysis of its carrier system in brush-border membrane vesicles of the guinea-pig ileum

Transport of l-proline was studied with membrane vesicles prepared from the brush borders of the guinea-pig ileum. The presence of an Na + gradient from outside to inside of the vesicles stimulated l-proline uptake. Accumulation of amino acid in the vesicles reached a maximum 30 s after incubation, then decreased due to efflux and finally equilibrated at a level nearly identical to that shown in the absence of an Na + gradient in 30 min. The peak level of the uptake was 3.5-times greater than the final equilibrium level. The equilibrium level of l-proline uptake decreased with increasing medium osmolarity. Extrapolation to infinite medium osmolarity, that is, under the condition of zero intravesicular space, showed no uptake, indicating transport of l-proline into membrane vesicles. The initial rate of uptake for 15 s was enhanced with increasing concentrations of Na + in the external medium. A small part of the l-proline transport occurred by simple diffusion in addition to Na +-gradient-dependent transport. When l-proline concentrations were varied and transport due to diffusion was subtracted, the initial rate of uptake dependent on Na + gradient (out > in) obeyed Michaelis-Menten kinetics with K m and V values of 0.67 mM and 2.73 nmol/15 s per mg protein, respectively. Evidence was obtained which indicates that l-cysteine is a substrate specific for transport through system ASC (alanine-, serine-, and cysteine-preferring) and that transport in the presence of an Li + gradient (out > in) also takes place by the ASC system. The uptake of l-proline in the presence of an Na + gradient (out > in) was inhibited 90% by a large excess of α-(methylamino)-isobutyrate, the model substrate specific for the A system (alanine-preferring). This indicates that 90% of Na +-gradient-dependent l-proline uptake is supported by the A system. The remaining 10% of l-proline uptake was found to be catalyzed by the ASC system, since l-proline uptake equivalent to this α-(methylamino)-isobutyrate-uninhibited part was demonstrated in the presence of an Li + gradient.

Biochemistry of L-proline-triggered germination of Bacillus megaterium spores.

The mechanism by which L-proline triggers germination in Bacillus megaterium QM B1551 spores was investigated. First, brief exposure of spores to L-proline, followed by dilution, was sufficient to trigger germination. Once germination was triggered, the spores continued initiation of germination and did not require high concentrations of L-proline. Triggering of germination was pH and temperature dependent. Second, enzymes for L-proline catabolism were absent in spores, and several non-metabolizable analogs of L-proline were effective trigger compounds. Third, triggering of germination occurred in the presence of inhibitors of proton motive force production, oxygen uptake, and metabolism. Fourth, uptake of L-proline occurred after the triggering of germination. These results argue that neither uptake nor metabolism of L-proline was necessary to trigger germination. Instead, L-proline probably causes a biophysical alteration in the spores that triggers the biochemical changes in germination.

Proline transport carrier-defective mutants of Escherichia coli K-12: properties and mapping

A series of mutants of Escherichia coli K-12 requiring a high concentration of L-proline for growth were isolated from a proline auxotroph strain, JE2133. Genetic studies of the mutants, PT19, PT21, and PT22, showed that all the mutations (proT) were point mutations, and these were mapped at 82 min on the E. coli genetic map. Intact cells and cytoplasmic membrane vesicles of these mutants were specifically defective in L-proline transport activity. Strain PT21 had no detectable activity of the L-proline transport carrier at all, and strains PT19 and PT22 had only 1/35 and 1/70, respectively, of the transport activity of the parental strain. The mutants were also shown to have a defect in proline-binding function of the carrier by measuring specific binding of proline to sonically disrupted membranes. These results indicate that the gene proT determines the function of proline carrier in the cytoplasmic membrane.

Carbon and Nitrogen Nutrition of Sclerotium cepivorum

SUMMARY Several monosaccharides (glucose, galactose, mannose, fructose), diand trisaccharides (cellobiose, sucrose, raffinose, maltose), and polysachcharides (inulin, starch, dextrin, glycogen) were excellent carbon sources for growth and production of sclerotia of Sclerotium cepivorum. Inulin and soluble starch were exceptionally good sources of carbon for production of sclerotia by several isolates of the fungus. Pectic substances, gluconic acid, and sorbitol were utilized slightly for growth. Several carbohydrate derivatives and Krebs cycle intermediates were not utilized. Casein hydrolysate, vitamin-free casamino acids, NaNO3, NH4CI, the amino acids isoleucine, arginine, proline, aspartic acid, leucine, and the amides asparagine and glutamine were the best nitrogen sources for growth. Valine, lysine, serine, glycine, histidine, and threonine were not utilized for growth. Proline, arginine, histidine, casein hydrolysate, and glutamine were the only nitrogen sources that allowed production of sclerotia by all isolates tested. Optimum temperature range for growth of S. cepivorum was 15-20 C, and the fungus grew better at 10 than at 25 C. The optimum concentration of carbon/liter from glucose depended on the isolate used. For one isolate, growth increased linearly with an increase of glucose carbon from 0 to 20.0 g/liter. Two g of nitrogen/liter from L-proline or NaNO3 was the optimum concentration for growth. High concentrations of L-proline in the medium (more than 0.25 g nitrogen/liter) supported excellent production of sclerotia by three isolates.

Amino acid transport by isolated mammalian renal tubules: III. Binding of l-proline by proximal tubule membranes

A particulate, brush border-rich membrane fraction was prepared from isolated rabbit renal tubules by agitation in 5 mM disodium EDTA and differential centrifugation. This membrane fraction bound l-proline. Binding was maximal during the first minute of incubation and reached equilibrium within 5 min. Saturation occurred at medium l-proline concentrations of 75 mM. Binding was inhibited completely by p- chloromercuribenzoate and by thermal denaturation. Deletion of Na + from the incubation medium reduced binding significantly as did NaCN. Ouabain and 2,4-dinitrophenol, however, had no inhibitory effect on binding. Glycine and l-alanine, known to share renal transport sites with proline, inhibited binding, but l-valine and l-phenylalanine did not. These results suggest that the observed binding process represents the initial step in the transtubular transport of l-proline.

AN OLPIDIUM PARASITE OF ALLOMYCES

The rate of germination of conidia of wildtype and mutant Neurospora crassa is a growth response that can be measured accurately and rapidly. A procedure is described by means of which convenient and reproducible measurements of germination can be made. The age of the conidia and their concentration proved to be critical factors in controlling the rate of germination. Under standard conditions germination of almost all of the conidia from a wild-type strain occurs in the period between 1-5 hr. The capacity to germinate at different times is randomly distributed among the conidia. This is also true for a proline-requiring mutant when grown on an optimal concentration of L-proline. On limiting concentrations of proline germination is slower, so that at any given time a linear relation can be obtained between the percentage of the conidia germinated and the logarithm of the concentration of proline. Similar relations exist between the germination of conidia from leucineless and lysineless mutants of Neurospora and their respective growth factors. The germination response is very rapid and sensitive to minute amounts of amino acid.