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PubMed1982

Nucleotide sequence of a spinach chloroplast proline tRNA.

The nucleotide sequence of a spinach chloroplast proline tRNA (sp. chl. tRNApro) has been determined. This tRNA shows more overall homology to phage T4 proline tRNA (61% homology) than to eukaryotic proline tRNAs (53% homology) or mitochondrial proline tRNAs (36-49% homology). Sp. chl. tRNApro, like all other chloroplast tRNAs sequenced, contains a methylated GG sequence in the dihyrouridine loop and lacks unusual structural features which have been found in many mitochondrial tRNAs.

Base Sequence↗
PubMed1985

Proline iminopeptidase from Bacillus coagulans: purification and enzymatic properties.

Proline iminopeptidase [EC 3.4.11.5] was purified about 2,700-fold from cell-free extract of Bacillus coagulans by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose, and hydroxyapatite, and gel filtration on Sephadex G-150. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.3 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid including proline, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminus. Pro-D-amino acid bonds were also susceptible to the enzyme. The enzyme was completely inhibited by p-chloromercuribenzoate (PCMB) and partially by proline but not by metal chelators, diisopropylphosphorofluoridate (DFP), or phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by incubation with 2-mercaptoethanol. These results and the chromatographic profile on PCMB-T-Sepharose suggest that the enzyme is a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0, and the molecular weight of the enzyme was estimated to be 40,000 by gel filtration on Sephadex G-100 and 35,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, indicating that the enzyme exists as a monomer.

Aminopeptidases↗
PubMed1970

Synthesis and Secretion of Hydroxyproline-containing Macromolecules in Carrots: II. In vivo Conversion of Peptidyl Proline to Peptidyl Hydroxyproline.

The chelator alpha,alpha'-dipyridyl prevents the formation of protein-bound hydroxyproline without affecting protein synthesis as measured by the incorporation of (14)C-proline. The inhibitory effect can be overcome by exogenous ferrous ions. Neither alpha,alpha'-dipyridyl nor Fe(2+) interferes in any other known way with the synthesis and secretion of the hydroxyproline-rich proteins normally found in the cell wall. This reversal by Fe(2+) of the inhibition of proline hydroxylation by alpha,alpha'-dipyridyl is used for a study in vivo of the hydroxylation reaction. This reaction has a temperature coefficient of 2.2, suggesting that it is an enzymatic process. Reversal by Fe(2+) of the alpha,alpha'-dipyridyl inhibition can occur after protein synthesis has been arrested with cycloheximide, indicating that peptidyl proline is the substrate of the hydroxylation reaction. Hydroxylation occurs in the cytoplasm, and not in the cell wall, and only for a limited time after the incorporation of proline into the polypeptide chain. This suggests a spatial separation of the enzyme and the substrate.

Journal Article↗
PubMed1979

Proline: A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L.

Proline is an effective cryoprotectant for the storage of cultured cells of Zea mays L. in liquid N(2). Increased freeze tolerance can be achieved by pregrowth for 3 to 4 days in medium containing proline. Cells cryoprotected with proline have an increased recovery potential when compared with cells cryoprotected with dimethylsulfoxide and glycerol. They also show a reduced postthaw viability loss and greater tolerance of a range of postthaw culture conditions. It is suggested that the mechanism of action of proline may be similar to that in its putative role of conferring protection against natural stresses. It may be protecting the cell against solution effects caused by dehydration during freezing. These findings are discussed in relation to other freeze tolerance enhancing treatments.

Journal Article↗
PubMed1981

Submitochondrial location and electron transport characteristics of enzymes involved in proline oxidation.

Isolated corn mitochondria (Zea mays cv. B73 x Mo17) were fractionated and the fragments were separated on a 20-45% (weight/weight) continuous sucrose gradient. Soluble enzymes remained at the top of the gradient overlapping with the outer membranes, while inner membrane vesicles and intact inner membranes were distributed farther down the gradient. Proline oxidase and Delta(1)-pyrroline-5-carboxylic acid dehydrogenase activities were associated only with the inner mitochondrial membrane. Glutamate dehydrogenase was confirmed as a matrix enzyme.Both proline and Delta(1)-pyrroline-5-carboxylic acid supported oxygen uptake in isolated mitochondria. Proline dependent oxygen uptake was relatively independent of pH with a maximum rate at pH 7.2. In contrast, Delta(1)-pyrroline-5-carboxylic acid-dependent oxygen uptake was sensitive to pH with an optimum at pH 6.1. The oxidation of proline and Delta(1)-pyrroline-5-carboxylic acid was inhibited by 10 micromolar rotenone. This indicates that electrons from these substrates enter the respiratory chain prior to at least one of the rotenone sensitive iron-sulfur proteins. Both substrates yielded ADP:O ratios of around 1.9 as compared to malate plus pyruvate (2.1), succinate (1.3), and exogenous NADH (1.2).

Journal Article↗
PubMed1983

Influence of Water Stress on the Vacuole/Extravacuole Distribution of Proline in Protoplasts of Nicotiana rustica.

Tobacco (Nicotiana rustica) plants were stressed by addition of polyethylene glycol solution (-20 bar) to the growth medium. The proline contents and concentrations in total protoplasts, vacuoles, and extravacuolar fractions of these plants have been determined and compared with protoplasts and cell fractions of well-watered plants. As compared to the control, the stress treatment of intact plants results in a 7-fold increase of the proline content in the extravacuolar fraction while the vacuolar content was enriched only 2.6-fold. In protoplasts of control plants, a proline concentration ratio (extravacuole to vacuole) of 1 was measured. In protoplasts of stressed plants, this ratio was nearly 3. Thus, water stress seems to have an effect on a tonoplast-localized transport system for proline.

Journal Article↗
PubMed1983

Translocation of carbohydrates and proline in young grapefruit trees at low temperatures.

Girdling and defoliation of fruit-bearing grapefruit (Citrus paradisi Macf.) branches inhibited the accumulation of soluble carbohydrates and proline in fruit tissues during low temperature treatment of trees. These treatments did not inhibit hydrolysis of sucrose to reducing sugars. Flavedo and albedo tissues responded similarly to low temperatures but little or no change occurred in the juice. Therefore, soluble carbohydrates and proline do not appear to interchange between different tissues of the fruit at low temperatures but instead are translocated into the fruit from other parts of the plant. Girdling fruit-bearing branches immediately after low temperature treatments inhibited the accumulation of sucrose in fruit tissues at dehardening temperatures. Also, proline levels decreased rapidly in fruit on girdled branches at dehardening temperatures. This rapid decrease suggests proline may serve as a source for respiratory energy in grapefruit during rapidly changing temperatures that favor active growth and during recovery of citrus from environmental stress.

Journal Article↗
PubMed1987

Characterization of Growth, Water Relations, and Proline Accumulation in Sodium Sulfate Tolerant Callus of Brassica napus L. cv Westar (Canola).

Unselected and sodium sulfate tolerant callus cultures of Brassica napus L. cv Westar were grown on media supplemented with mannitol, NaCl, or Na(2)SO(4). In all cases, growth of tolerant callus, measured on a fresh weight or dry weight basis, was greater than that of unselected callus, which was also subject to necrosis on high levels of salt. Tissue water potential became more negative in both unselected and tolerant callus grown in the presence of mannitol or Na(2)SO(4). Water potentials in unselected callus were more negative than those of the tolerant tissues; but over a range of Na(2)SO(4) concentrations both cultures displayed osmotic adjustment, maintaining relatively constant turgor. Proline accumulation in both unselected and tolerant callus was low (15 to 20 micromoles per gram dry weight) in the absence of stress, but increased on media supplemented with mannitol, NaCl, or Na(2)SO(4). Increases in proline concentration were approximately linear in tolerant callus, reaching a maximum of 130 to 175 micromoles per gram dry weight. In unselected callus, concentrations were higher, reaching 390 to 520 micromoles per gram dry weight. Proline accumulation was correlated with inhibition of growth, and there was a negative correlation between proline concentration and culture age for tolerant callus.

Journal Article↗
PubMed1989

Oxidation of Proline and Glutamate by Mitochondria of the Inflorescence of Voodoo Lily (Sauromatum guttatum).

In appendices of Sauromatum guttatum that are developing thermogenicity, mitochondria isolated from successive developmental stages of the inflorescence show an increase in the oxidation rates of proline and glutamate. A similar rise in the oxidation rates of these compounds is observed in mitochondria obtained from the spathe, a nonthermogenic organ of the inflorescence. Changes in oxidative metabolism were also observed in mitochondria isolated from sections of immature appendix treated with salicylic acid (SA) at 0.69 microgram per gram fresh weight indicating that they are induced by SA. At that concentration, however, SA has no effect on oxygen consumption by mitochondria in the presence of glutamate, proline, or malate. Furthermore, oxygen uptake by mitochondria in the presence of proline or glutamate is partially sensitive to salicylhydroxamic acid (SHAM) at concentrations greater than 2 millimolar when in the presence of 1 millimolar KCN. For NADH, succinate, and malate a high capacity of the alternative (cyanide-resistant) pathway is found that is completely sensitive to SHAM at 1.5 to 4 millimolar. The increase in the mitochondrial capacity to oxidize either amino acid is also found in four other Araceae species including both thermogenic and nonthermogenic ones. After anthesis, the rates of proline and glutamate oxidation decline.

Journal Article↗
PubMed1991

Peptidyl proline hydroxylation and the growth of a soybean cell culture.

Peptidyl proline hydroxylase inhibitors block the growth of cultured soybean (Glycine max) cells and bring about the disappearance of the major salt-extractable hydroxyproline-rich protein, the 33 kilodalton repetitive proline-rich protein (RPRP2). Three polypeptides of 28, 20, and 14 kilodalton that cross-react with an antibody to RPRP2 accumulate in the culture during steady-state growth. In the presence of the proline hydroxylase inhibitors, all of these repetitive proline-rich proteins disappear. These results indicate that the hydroxyproline-rich proteins play a role in cell growth, and that hydroxylation may regulate the steady-state level of at least one of these proteins by influencing its turnover.

Journal Article↗
PubMed1992

Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures.

The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy-dependent programs.

Journal Article↗
PubMed1999

Glutamine synthetase in the phloem plays a major role in controlling proline production

To inhibit expression specifically in the phloem, a 274-bp fragment of a cDNA (Gln1-5) encoding cytosolic glutamine synthetase (GS1) from tobacco was placed in the antisense orientation downstream of the cytosolic Cu/Zn superoxide dismutase promoter of Nicotiana plumbaginifolia. After Agrobacterium-mediated transformation, two transgenic N. tabacum lines exhibiting reduced levels of GS1 mRNA and GS activity in midribs, stems, and roots were obtained. Immunogold labeling experiments allowed us to verify that the GS protein content was markedly decreased in the phloem companion cells of transformed plants. Moreover, a general decrease in proline content in the transgenic plants in comparison with wild-type tobacco was observed when plants were forced to assimilate large amounts of ammonium. In contrast, no major changes in the concentration of amino acids used for nitrogen transport were apparent. A (15)NH(4)(+)-labeling kinetic over a 48-hr period confirmed that in leaves of transgenic plants, the decrease in proline production was directly related to glutamine availability. After 2 weeks of salt treatment, the transgenic plants had a pronounced stress phenotype, consisting of wilting and bleaching in the older leaves. We conclude that GS in the phloem plays a major role in regulating proline production consistent with the function of proline as a nitrogen source and as a key metabolite synthesized in response to water stress.

Journal Article↗
PubMed1998

Crystallization and preliminary X-ray analysis of human platelet profilin complexed with an oligo proline peptide.

Profilin is an actin-monomer binding protein that regulates the distribution and dynamics of the actin cytoskeleton. Profilin binds poly-L-proline and proline-rich peptides in vitro and co-localizes with proline-rich proteins in focal adhesions and at the site of actin tail assembly on the surface of intracellular parasites such as Listeria monocytogenes. The crystallization of the complex between human platelet profilin (HPP) and an L-proline decamer [(Pro)10] is reported here. Diffraction from these crystals is consistent with the space group P21212 with unit-cell constants a = 68.25, b = 97.64, c = 39.10 A. The crystals contain two HPP molecules per asymmetric unit and diffract to 2.2 A.

Blood Platelets↗
PubMed1993

Molecular cloning and characterization of a proline iminopeptidase gene from Neisseria gonorrhoeae.

Proline iminopeptidase (Pip) is a hydrolase elaborated by virtually all strains of Neisseria gonorrhoeae that selectively removes N-terminal proline residues from peptides. Escherichia coli clones expressing the gonococcal gene coding for Pip were identified in a genomic cosmid library using a synthetic colorimetric substrate. Nucleotide sequence determination and analyses of polypeptides detected by coupled in vitro transcription/translation reactions revealed that Pip is a 311-amino-acid polypeptide with a M(r) of 35 kDa and a pI of 5.4. Southern hybridization showed that the pip gene is present in a single copy on the chromosome of N. gonorrhoeae strain MS11 which maps immediately upstream of the previously identified opaA locus. The transcriptional start site of pip in E. coli, determined by primer extension analysis, was characteristic of an NtrA or sigma-54-dependent promotor. Complementation of an E. coli mutant deficient in both proline biosynthesis and dipeptide uptake confirmed that Pip is capable of releasing biologically active proline from peptides. Pip expression was found to be non-essential for in vitro growth of N. gonorrhoeae, based on the viability of a Pip- gonococcal mutant.

Amino Acid Sequence↗
PubMed1997

Synthesis and NMR spectroscopy of peptides containing either phosphorylated or phosphonylated cis- or trans-4-hydroxy-L-proline.

Many proteins are regulated by reversible O-glycosylation and O-phosphorylation. Whereas O-glycosylation of hydroxy-L-proline is common and well investigated, phosphorylation has not been proved so far in vivo, but this post-translational modification is entirely possible. As a first step to identify this phosphoamino acid, we describe both the syntheses of peptides phosphorylated at 4-hydroxy-L-proline and the 1H and 31P NMR parameters of these phosphopeptides. The model peptides were synthesized on solid-phase using Fmoc-strategy. Both natural isomers of 4-hydroxy-L-proline (containing the hydroxyl group in either the cis or trans position) were introduced without side-chain protection. All peptides were globally phosphorylated with O,O'-tert-butyl-N,N-diethylphosphoramidite on the solid phase and cleaved with trifluoroacetic acid. Additionally, we synthesized two classes of phosphonopeptides that mimic phosphopeptides, namely H- and methylphosphonopeptides. The NMR data were based on the model peptide Gly-Gly-Hyp-Ala, which is regarded as a typical random-coil sequence. The NMR parameters showed a significant influence of the phosphate group on the cis-trans isomerization of the Gly-Hyp bond, which may reflect a possible regulation of proteins by changing their local conformations. The 1H and 31P NMR parameters differed for each isomer, and were distinct from the parameters of phosphorylated serine, threonine and tyrosine. These known shifts can be used to identify both cis- and trans-O-phospho-4-hydroxy-L-proline in vivo.

Chromatography, High Pressure Liquid↗
PubMed1999

Purification and characterization of an extracellular proline iminopeptidase from Arthrobacter nicotianae 9458.

An extracellular proline iminopeptidase, with a molecular mass of about 53 kDa, was purified from Arthrobacter nicotianae 9458 and characterized. The enzyme had temperature and pH optima of 37 degrees C and 8.0, respectively, was completely inactivated by heating for 1 min at 80 degrees C and showed highest activity on Pro-pNA. The proline iminopeptidase was characterized by activity at low temperature, NaCl concentrations up to 7.5% and by high sensitivity to pH values 6.0, serine enzyme inhibitor PMSF and divalent cations, Fe2+, Sn2+, Cu2+, Zn2+, Hg2+, Co2+ and Ni2+. The extracellular proline iminopeptidase from A. nicotianae 9458 was able to hydrolyze proline-containing peptides at the pH, temperature and NaCl concentration typical of the surface of smear-ripened cheese and may contribute to proteolysis of these cheeses during ripening.

Aminopeptidases↗
PubMed2000

PNRC: a proline-rich nuclear receptor coregulatory protein that modulates transcriptional activation of multiple nuclear receptors including orphan receptors SF1 (steroidogenic factor 1) and ERRalpha1 (estrogen related receptor alpha-1).

PNRC (proline-rich nuclear receptor coregulatory protein) was identified using bovine SF1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC is unique in that it has a molecular mass of 35 kDa, significantly smaller than most of the coregulatory proteins reported so far, and it is proline-rich. PNRC's nuclear localization was demonstrated by immunofluorescence and Western blot analyses. In the yeast two-hybrid assays, PNRC interacted with the orphan receptors SF1 and ERRalpha1 in a ligand-independent manner. PNRC was also found to interact with the ligand-binding domains of all the nuclear receptors tested including estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), and retinoid X receptor (RXR) in a ligand-dependent manner. Functional AF2 domain is required for nuclear receptors to bind to PNRC. Furthermore, in vitro glutathione-S-transferase pull-down assay was performed to demonstrate a direct contact between PNRC and nuclear receptors such as SF1. Coimmunoprecipitation experiment using Hela cells that express PNRC and ER was performed to confirm the interaction of PNRC and nuclear receptors in vivo in a ligand-dependent manner. PNRC was found to function as a coactivator to enhance the transcriptional activation mediated by SF1, ERR1 (estrogen related receptor alpha-1), PR, and TR. By examining a series of deletion mutants of PNRC using the yeast two-hybrid assay, a 23-amino acid (aa) sequence in the carboxy-terminal region, aa 278-300, was shown to be critical and sufficient for the interaction with nuclear receptors. This region is proline rich and contains a SH3-binding motif, S-D-P-P-S-P-S. Results from the mutagenesis study demonstrated that the two conserved proline (P) residues in this motif are crucial for PNRC to interact with the nuclear receptors. The exact 23-amino acid sequence was also found in another protein isolated from the same yeast two-hybrid screening study. These two proteins belong to a new family of nuclear receptor coregulatory proteins.

Amino Acid Motifs↗
PubMed1998

Proline powers pre-flight warm-up in the african fruit beetle pachnoda sinuata

We investigated thoracic temperatures (Tth) during different activities of the endothermic fruit beetle Pachnoda sinuata and analysed which energy substrates are used for the pre-flight warm-up of its flight muscles. Pachnoda sinuata elevates its Tth prior to take-off either by basking in the sun or by warming endothermically to a narrow range around 34 degreesC. During lift-generating tethered flight at low ambient temperatures (Ta=25 degreesC), Tth of P. sinuata decreases steadily until it reaches 28 degreesC, which is not sufficiently high to sustain flight. Tth remains stable during lift-generating tethered flight at high Ta (31 degreesC). Wingbeat frequency (fw) is dependent on Tth: when Tth declines, fw decreases in a linear manner. The proline concentrations in the haemolymph and flight muscles decrease during warm-up. In contrast, the carbohydrate levels in the haemolymph and flight muscles are not affected by the warm-up process, while the glycogen level of the flight muscles declines significantly during the first 10 s of lift-generating tethered flight. This suggests that the energy for endothermic warm-up is produced solely by the oxidation of proline. Measurements of the respiratory quotient (RQ) confirmed that P. sinuata uses a combination of carbohydrates and proline to power its flight. At rest and during lift-generating tethered flight, the RQ is approximately 0.9. During warm-up, the RQ is significantly lower at 0.82, which is close to the theoretical value of 0.8 for the partial oxidation of proline. The rate of oxygen consumption during endothermic warm-up is 45 % of that during lift-generating tethered flight

Journal Article↗