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At least 433 records · Page 24Linked to original sources
PubMed1988

Serum proline and blood lactate levels in alcoholic patients without hepatic failure: relationship with alcohol ingestion and histological activity.

It has not yet been established whether serum proline and blood lactate levels are increased in alcoholic liver disease. We measured serum proline and blood lactate in controls and in patients with different stages of alcoholic liver disease in the absence of hepatic failure. Samplings were done in both abstinent and drinking alcoholics. Compared to controls, there was a striking increase in serum proline levels in 52 abstinent alcoholics with little or no hepatic fibrosis by histological assessment (0.10 +/- 0.01 vs. 0.155 +/- 0.008; p less than 0.005). Blood lactate levels were within the normal range and did not correlate with serum proline levels. On the other hand, serum proline and blood lactate levels were independent of hepatic necrosis and inflammation scores. In addition, in 10 patients with blood alcohol concentrations between 0.3 mg/ml and 7.8 mg/ml, serum lactate and proline were significantly elevated (2.42 +/- 0.29 mg/ml and 0.275 +/- 0.0026 mg/ml, respectively; p less than 0.005). These results show that there is an association between serum proline levels and the abstinence period in alcoholic patients. They further suggest that in alcoholic patients neither serum proline nor blood lactate concentrations are reliable markers for liver histological activity (necrosis and inflammation) or fibrosis.

Adult↗
PubMed1984

Proline transport increases growth efficiency in salt-stressed Streptomyces griseus.

Streptomyces griseus synthesizes proline for osmoregulation under salt stress. Uptake of exogenous [14C]proline and internal synthesis of proline were quantified in cells growing at salt concentrations from 0 to 1 M NaCl. Externally supplied proline accounted for an increased proportion of the intracellular pool of free proline as salt concentration was increased, but neither the concentration nor the composition of the internal amino acid pool was substantially altered by supply of exogenous proline. Uptake of exogenous proline significantly increased the specific growth yield of S. griseus growing under salt stress; the increased yield was proportional to reductions in proline synthesis.

Biological Transport↗
PubMed1998

Construction of a proline-producing mutant of the extremely thermophilic eubacterium Thermus thermophilus HB27.

Growth of Thermus thermophilus HB27 was inhibited by a proline analog, 3,4-dehydroproline (DHP). This result suggested that the gamma-glutamyl kinase (the product of the proB gene) was inhibited by feedback inhibition in T. thermophilus. DHP-resistant mutants were reported previously for Escherichia coli (A. M. Dandekar and S. L. Uratsu, J. Bacteriol. 170:5943-5945, 1988) and Serratia marcescens (K. Omori, S. Suzuki, Y. Imai, and S. Komatsubara, J. Gen. Microbiol. 138:693-699, 1992), and their mutated sites in the proB gene were identified. Comparison of the amino acid sequence of T. thermophilus gamma-glutamyl kinase with those of E. coli and S. marcescens mutants revealed that the DHP resistance mutations occurred in the amino acids conserved among the three organisms. For eliminating the feedback inhibition, we first constructed a DHP-resistant mutant, TH401, by site-directed mutagenesis at the proB gene as reported for the proline-producing mutant of S. marcescens. The mutant, TH401, excreted about 1 mg of L-proline per liter at 70 degreesC after 12 h of incubation. It was also suggested that T. thermophilus had a proline degradation and transport pathway since it was able to grow in minimal medium containing L-proline as sole nitrogen source. In order to disrupt the proline degradation or transport genes, TH401 was mutated by UV irradiation. Seven mutants unable to utilize L-proline for their growth were isolated. One of the mutants, TH4017, excreted about 2 mg of L-proline per liter in minimal medium at 70 degreesC after 12 h of incubation.

Amino Acid Sequence↗
PubMed2001

Mutations in the listerial proB gene leading to proline overproduction: effects on salt tolerance and murine infection.

The observed sensitivity of Listeria monocytogenes to the toxic proline analogue L-azetidine-2-carboxylic acid (AZ) suggested that proline synthesis in Listeria may be regulated by feedback inhibition of gamma-glutamyl kinase (GK), the first enzyme of the proline biosynthesis pathway, encoded by the proB gene. Taking advantage of the Epicurian coli mutator strain XL1-Red, we performed random mutagenesis of the recently described proBA operon and generated three independent mutations in the listerial proB homologue, leading to proline overproduction and salt tolerance when expressed in an E. coli (DeltaproBA) background. While each of the mutations (located within a conserved 26-amino-acid region of GK) was shown to confer AZ resistance (AZ(r)) on an L. monocytogenes proBA mutant, listerial transformants failed to exhibit the salt-tolerant phenotype observed in E. coli. Since proline accumulation has previously been linked to the virulence potential of a number of pathogenic bacteria, we analyzed the effect of proline overproduction on Listeria pathogenesis. However, our results suggest that as previously described for proline auxotrophy, proline hyperproduction has no apparent impact on the virulence potential of Listeria.

Amino Acid Sequence↗
PubMed1988

Proline incorporation into protein by Rickettsia prowazekii during growth in Chinese hamster ovary (CHO-K1) cells.

Both the requirement of Rickettsia prowazekii for the amino acid proline for growth and rickettsial proline incorporation were determined in Chinese hamster ovary (CHO-K1) cells auxotrophic for proline. Incubation of cells in Dulbecco modified Eagle medium supplemented with various concentrations of proline resulted in a range of host intracellular proline pools, as determined by both dansylation and equilibration of specific radioactivities. Maximal rickettsial growth was observed only in host cells with an intracellular proline pool of 1.0 mM or greater. Protein synthesis by rickettsiae in infected cells was determined to be the difference between emetine-resistant proline incorporation in the presence and absence of chloramphenicol. After density gradient centrifugation in Percoll, a rickettsial band with associated radioactivity was observed in lysates of infected cells treated with emetine but not in lysates of infected cells treated with both emetine and chloramphenicol. The average amount of proline incorporated into protein in situ was determined to be 6.3 +/- 0.8 amol per rickettsia. These results, obtained with a system which allows the study of rickettsiae in their natural habitat, are discussed in light of existing information about protein synthesis in isolated rickettsiae.

Animals↗
PubMed1969

Reactions involved in the conversion of ornithine to proline in Clostridia.

An enzyme system which converts ornithine to proline was partially purified from extracts of cells of Clostridium botulinum and of Clostridium PA 3670 by fractionation with ammonium sulfate and by dialysis in the presence of 0.01 m ornithine. Nicotinamide adenine dinucleotide (NAD) was the only cofactor required for maximal activity of the partially purified system. A possible intermediate in the conversion was accumulated when a high concentration of proline was used as substrate and the NAD was maintained in the oxidized state by adding lactic dehydrogenase. Small but significant amounts of this or a similar compound were trapped with either ornithine or proline as substrate when o-aminobenzaldehyde was added to reaction mixtures. The accumulation of the o-aminobenzaldehyde reaction product was NAD-dependent with both substrates. The compound accumulated from proline was identified as Delta(1)-pyrroline-5-carboxylic acid on the basis of the melting point of the 2,4-dinitrophenylhydrazone, and by paper chromatography of the reaction product formed with o-aminobenzaldehyde. Also, extracts of C. botulinum cells oxidized reduced NAD (NADH) in the presence of the product from proline or in the presence of Delta(1)-pyrroline-5-carboxylic acid, but did not do so in the presence of the other possible intermediate, Delta(1)-pyrroline-2-carboxylic acid. (14)C-Delta(1)-pyrroline-5-carboxylic acid was reduced to (14)C-proline by these extracts in the presence of NADH. These data indicate that the conversion of ornithine to proline by C. botulinum and Clostridium PA 3679 cells involves an oxidation of ornithine to glutamic-gamma-semialdehyde which undergoes ring closure to Delta(1)-pyrroline-5-carboxylic acid. The latter compound is then reduced to proline.

Carbon Isotopes↗
PubMed1979

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.

Bacillus megaterium↗
PubMed1981

Proline transport in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is capable of utilizing proline as the sole source of nitrogen. Mutants of S. cerevisiae with defective proline transport were isolated by selecting for resistance to either of the toxic proline analogs L-azetidine-2-carboxylate or 3,4-dehydro-DL-proline. Strains carrying the put4 mutation are defective in the high-affinity proline transport system. These mutants could still grow when given high concentrations of proline, due to the operation of low-affinity systems whose existence as confirmed by kinetic studies. Both systems were repressed by ammonium ions, and either was induce by proline. Low-affinity transport was inhibited by histidine, so put4 mutants were unable to grow on a medium containing high concentrations of proline to which histidine has been added.

Amino Acids↗
PubMed2004

The chemical chaperone proline relieves the thermosensitivity of a dnaK deletion mutant at 42 degrees C.

Since, like other osmolytes, proline can act as a protein stabilizer, we investigated the thermoprotectant properties of proline in vitro and in vivo. In vivo, elevated proline pools in Escherichia coli (obtained by altering the feedback inhibition by proline of gamma-glutamylkinase, the first enzyme of the proline biosynthesis pathway) restore the viability of a dnaK-deficient mutant at 42 degrees C, suggesting that proline can act as a thermoprotectant for E. coli cells. Furthermore, analysis of aggregated proteins in the dnaK-deficient strain at 42 degrees C by two-dimensional gel electrophoresis shows that high proline pools reduce the protein aggregation defect of the dnaK-deficient strain. In vitro, like other "chemical chaperones," and like the DnaK chaperone, proline protects citrate synthase against thermodenaturation and stimulates citrate synthase renaturation after urea denaturation. These results show that a protein aggregation defect can be compensated for by a single mutation in an amino acid biosynthetic pathway and that an ubiquitously producible chemical chaperone can compensate for a defect in one of the major chaperones involved in protein folding and aggregation.

Citrate (si)-Synthase↗
PubMed1966

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.

Antimetabolites↗
PubMed1968

Proline as an intermediate in the reductive deamination of ornithine to delta-aminovaleric acid.

Fresh extracts of cells of Clostridium botulinum reduced a limited amount of ornithine to delta-aminovaleric acid, but at high substrate concentrations a considerable amount of an amino compound accumulated which was neutral at pH 4.2. Aging of the extracts at -10 C or freezing and thawing resulted in the loss of the ability to produce delta-aminovaleric acid, but the ability to produce the neutral compound was retained. This compound was separated by column chromatography, and was found to be identical to dl-proline with respect to (i) R(F) upon paper chromatography, (ii) migration rates upon paper ionophoresis, (iii) spectrum of the product of the ninhydrin reaction, (iv) oxidation with d-amino acid oxidase, and (v) rate of reduction to delta-aminovaleric acid by cell extracts. The intermediate role of proline in the reduction of ornithine to delta-aminovaleric acid was indicated by (i) rate studies with and without an added electron donor and with and without inhibitors of proline reductase, (ii) the initial accumulation of radioactive proline to the exclusion of radioactive delta-aminovaleric acid from (14)C-l-ornithine in the presence of low levels of carrier proline, and (iii) the initial accumulation of proline at low levels prior to a significant accumulation of delta-aminovaleric acid in reaction mixtures in which the latter compound was the primary product after a longer incubation time. The conversion of ornithine to proline was the rate-limiting step in the presence of a good electron donor (alanine). The mechanism of the conversion of ornithine to proline has not been established. Preliminary data indicated that it may involve an oxidation to glutamic-gamma-semialdehyde and its equilibrium product, Delta(1)-pyrroline-5-carboxylic acid.

Adenine Nucleotides↗
PubMed2000

The central proline of an internal viral fusion peptide serves two important roles.

The fusion peptide of the avian sarcoma/leukosis virus (ASLV) envelope protein (Env) is internal, near the N terminus of its transmembrane (TM) subunit. As for most internal viral fusion peptides, there is a proline near the center of this sequence. Robson-Garnier structure predictions of the ASLV fusion peptide and immediate surrounding sequences indicate a region of order (beta-sheet), a tight reverse turn containing the proline, and a second region of order (alpha-helix). Similar motifs (order, turn or loop, order) are predicted for other internal fusion peptides. In this study, we made and analyzed 12 Env proteins with substitutions for the central proline of the fusion peptide. Env proteins were expressed in 293T cells and in murine leukemia virus pseudotyped virions. We found the following. (i) All mutant Envs form trimers, but when the bulky hydrophobic residues phenylalanine or leucine are substituted for proline, trimerization is weakened. (ii) Surprisingly, the proline is required for maximal processing of the Env precursor into its surface and TM subunits; the amount of processing correlates linearly with the propensity of the substituted residue to be found in a reverse turn. (iii) Nonetheless, proteolytically processed forms of all Envs are preferentially incorporated into pseudotyped virions. (iv) All Envs bind receptor with affinity greater than or equal to wild-type affinity. (v) Residues that support high infectivity cluster with proline at intermediate hydrophobicity. Infectivity is not supported by mutant Envs in which charged residues are substituted for proline, nor is it supported by the trimerization-defective phenylalanine and leucine mutants. Our findings suggest that the central proline in the ASLV fusion peptide is important for the formation of the native (metastable) Env structure as well as for membrane interactions that lead to fusion.

Alpharetrovirus↗
PubMed2003

The proline repeat domain of p53 binds directly to the transcriptional coactivator p300 and allosterically controls DNA-dependent acetylation of p53.

The transcription coactivator p300 cannot acetylate native p53 tetramers, thus revealing intrinsic conformational constraints on p300-catalyzed acetylation. Consensus site DNA is an allosteric effector that promotes acetylation of p53, suggesting that p300 has an undefined conformationally flexible interface within the p53 tetramer. To identify such conformationally responsive p300-binding sites, p300 was subjected to peptide selection from a phage-peptide display library, a technique that can define novel protein-protein interfaces. The enriched p300-binding peptides contained a proline repeat (PXXP/PXPXP) motif, and five proline repeat motifs actually reside within the p53 transactivation domain, suggesting that this region of p53 may harbor the second p300 contact site. p300 binds in vitro to PXXP-containing peptides derived from the proline repeat domain, and PXXP-containing peptides inhibit sequence-specific DNA-dependent acetylation of p53, indicating that p300 docking to both the LXXLL and contiguous PXXP motif in p53 is required for p53 acetylation. Deletion of the proline repeat motif of p53 prevents DNA-dependent acetylation of p53 by occluding p300 from the p53-DNA complex. Sequence-specific DNA places an absolute requirement for the proline repeat domain to drive p53 acetylation in vivo. Chromatin immunoprecipitation was used to show that the proline repeat deletion mutant p53 is bound to the p21 promoter in vivo, but it is not acetylated, indicating that proline-directed acetylation of p53 is a post-DNA binding event. The PXXP repeat expands the basic interface of a p300-targeted transactivation domain, and proline-directed acetylation of p53 at promoters indicates that p300-mediated acetylation can be highly constrained by substrate conformation in vivo.

Acetylation↗
PubMed1988

Na-dependent L-proline transport by eel intestinal brush-border membrane vesicles.

L-[3H]proline uptake by brush-border membrane vesicles prepared from intestinal mucosa of the European eel, Anguilla anguilla, was stimulated by a transmembrane Na gradient (out greater than in). Kinetic analysis of L-proline influx, under short-circuited membrane potential conditions, indicated the presence of an apparent single Na-dependent carrier process (Kapp = 0.23 +/- 0.04 mM and Jmax = 7.96 +/- 0.87 nmol.mg protein-1.min-1) and a nonsaturable transfer component with an apparent diffusional permeability (P) of 1.53 +/- 0.35 microliter.mg protein-1.min-1. An imposed transmembrane potential (inside negative) increased apparent L-proline binding affinity (lowered Kapp) without appreciably altering maximal amino acid influx (Jmax). Hill analysis of L-proline influx over a wide range of external Na concentrations indicated a 1:1 stoichiometry for Na-proline cotransport. Use of amino acid inhibitors of L-proline influx suggested that L-proline transfer may occur by either a classical Na-dependent A System with a wide substrate specificity or by the combination of Na-dependent PHE (phenylalanine preferring) and IMINO (proline, alpha-methylaminoisobutyric acid preferring) Systems.

Anguilla↗
PubMed1990

Proline transport by brush-border membrane vesicles of lobster antennal glands.

Purified brush-border membrane vesicles (BBMV) of lobster antennal gland labyrinth and bladder were separately formed by a magnesium precipitation technique. L-[3H]proline uptake was stimulated by a transmembrane NaCl gradient [outside (o) greater than inside (i)] to a greater extent in BBMV from labyrinth than those from the bladder. Detailed study of the labyrinth proline-transport processes revealed a specific dependence on NaCl, with negligible stimulatory effects by NaSCN, Na-gluconate, or KCl. A transmembrane proton gradient (o greater than i) was without stimulatory effect on proline transport. A transmembrane potential difference alone, in the presence of equilibrated NaCl and L-[3H]proline, led to net influx of the labeled amino acid, suggesting that the uptake process was electrogenic and capable of bringing about the net transfer of positive charge to the vesicle interior. Although a transmembrane Na gradient alone, in the presence of equilibrated Cl and L-[3H]proline, was able to bring about the net influx of the amino acid, a transmembrane Cl gradient alone under Na- and L-[3H]proline-equilibrated conditions was not, suggesting that only the Na gradient could energize the carrier process through cotransport, while the anion served an essential activating role. Proline influx by these vesicles occurred by the combination of at least one saturable Michaelis-Menten carrier system (apparent Kt = 0.37 mM; apparent JM = 1.19 nmol.mg protein-1.10 s-1) and apparent diffusion (P = 0.33 nmol.mg protein-1.10 s-1.mM-1). Static head analysis of the transport process suggested a cotransport stoichiometry of 2 Na:1 proline with essential activation by Cl ion.

Amino Acids↗
PubMedDate not supplied

The role of the imino transporter protein in sepsis-impaired intestinal proline absorption.

Recently, sepsis has been shown to impair intestinal amino acid absorption in addition to gut metabolic and barrier functions. We investigated intestinal proline absorption in a rabbit model of sepsis. Twelve hours after intraperitoneal injection of lipopolysaccharide, proline uptake by everted jejunal sacs prepared from septic animals (480.4 +/- 67.4 nmol per sac per hour) was significantly reduced compared with controls (846.8 +/- 73.5 nmol per sac per hour) (p < .001 by t test). We next investigated whether reduced expression of transporter proteins contributed to the impaired intestinal proline uptake during sepsis. The proline (imino) carrier of rabbit jejunum is covalently bound by fluorescein isothiocyanate (FITC) and/or phenylisothiocyanate with irreversible inhibition of proline uptake. This binding and inhibition is prevented by sodium chloride and L-proline. Single-cell suspensions of rabbit enterocytes were prepared 12 hours after intraperitoneal injection of lipopolysaccharide/saline or saline alone. Enterocytes were incubated for 30 minutes in tris(hydroxymethyl)aminomethane/ethylenediaminetetraacetate (Tris/EDTA) buffer; buffer with 1 mM phenylisothiocyanate; or buffer with 10 mM proline, 100 mM sodium chloride, and 1 mM phenylisothiocyanate. After incubation with 10 microM FITC in Tris/EDTA buffer for 15 minutes, the percent positivity and fluorescent intensity of FITC binding to enterocytes were determined by using flow cytometry. Sepsis significantly reduced the percentage of enterocytes binding FITC and the fluorescent intensity of FITC binding of proline/sodium chloride-pretreated or untreated cells. This suggests that sepsis depresses the expression of imino transporters by rabbit enterocytes, which may explain the reduced intestinal proline absorption.

Amino Acid Transport Systems, Neutral↗
PubMedDate not supplied

Effects of L-proline on phase I and phase II xenobiotic biotransformation capacities of rat and human hepatocytes in long-term collagen gel cultures.

L-Proline supplementation of the medium for collagen gel cultures of hepatocytes has been shown to improve albumin secretion. A study was made as to whether L-proline is also essential for the maintenance of xenobiotic biotransformation capacities in collagen gel sandwich and immobilisation cultures of rat and human hepatocytes. Key phase I (cytochrome P450-dependent monooxygenase [CYP)] and microsomal epoxide hydrase [mEH]) and phase II (glutathione S-transferase [GST]) biotransformation enzyme activities and the secretion of albumin in the culture medium were assessed in the absence and presence of L-proline. CYP and mEH activities were not affected by the addition of L-proline, whereas phase II alpha-Class GST activity of rat hepatocytes in collagen cultures was decreased. Species differences were demonstrated, as human hepatocytes showed a better maintenance of GST activities than their rat counterparts in the presence of L-proline. Albumin secretion, often considered to be a marker for differentiated cell function, does not parallel the biotransformation capacities of the hepatocytes in culture. Additional results demonstrated an L-proline-mediated enhancement of the proliferation rate of contaminating stellate cells in conventional monolayer culture. Transdifferentiation of stellate cells to proliferating myofibroblasts, along with an increased albumin secretion and collagen synthesis, are characteristic of fibrotic liver. Since the last two phenomena have been observed in L-proline-supplemented collagen gel cultures, it can be concluded that when stable collagen gel cultures of rat hepatocytes are needed for long-term pharmacotoxicological studies, it is preferable to use an L-proline-free culture medium. Further studies on medium optimisation are required for hepatocytes from species other than rat.

Adult↗
PubMed1991

A proline residue near the amino terminus of the mature domain of secretory proteins lowers the level of the proton motive force required for translocation.

A large variety of proOmpF-Lpps, hybrid secretory proteins composed of the signal region of proOmpF and the mature part of the major lipoprotein, either possessing or not possessing a proline residue near the amino terminus of their mature domains, were constructed at a DNA level, and the rates of their in vitro translocation were determined in the presence and absence of the proton motive force (delta muH+). A proline residue at the signal peptide cleavage site (position +1) blocked the cleavage reaction but not the translocation reaction. All the proOmpF-Lpps examined exhibited approximately the same translocation rate in the presence of delta muH+ irrespective of the presence or absence of a proline residue near the amino terminus. In the absence of the delta muH+, which was achieved by either depletion of the respiratory substrate or the use of urea-treated membrane vesicles permeable to protons, proOmpF-Lpps possessing a proline residue near the amino terminus of the mature domain were translocated whereas those possessing no proline residue in this region were not translocated at all or only very weakly. The position of the proline residue was then moved stepwise away from the amino terminus of the mature domain. The further the position was moved away, the slower was the rate of translocation in the absence of delta muH+. The removal of the proline residue at position +2 of the mature domain of proOmpA also made the delta mu(H+)-independent translocation appreciably slower. It is suggested that the conformational flexibility endowed by the proline residue on the junction region between the signal peptide and the mature domain allows the translocation in the absence of delta muH+ and that this junction region must take on a particular conformation for initiation of the translocation reaction.

Amino Acid Sequence↗