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PubMed1991

Cloning and expression in Escherichia coli of a gene encoding proline oxidase of Serratia marcescens.

Proline plays a central role in the biosynthesis of prodigiosin by Serratia marcescens. Proline catabolism takes place by oxidation catalysed by the enzyme proline oxidase encoded by the gene putA. A gene bank of chromosomal DNA from S. marcescens was constructed using the plasmid vector pBR328, and then recombinant DNA was used in transformation experiments with Escherichia coli HB 101 as recipient strain. One of the recombinant plasmids, pSL001, was encoded for proline oxidase. Subcloning experiments led to a second plasmid pSL008 able to maintain proline oxidase activity.

Cloning, Molecular↗
PubMed1982

Study of a proline-rich polypeptide bound to the prostatic binding protein of rat ventral prostate.

A proline-rich polypeptide is associated with prostatic binding protein, a major androgen-dependent protein described previously in the rat ventral prostate. This polypeptide has been purified. Its molecular weight estimated by gel filtration is about 8500, but a markedly lower value (3300) is obtained by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. Isoelectric focusing on thin layer polyacrylamide gels yields two major forms with isoelectric points of, respectively, 7.75 and 7.05. The amino acid composition of proline-rich polypeptide is characterized by a high (19.5%) proline content and its NH2-terminal amino acid is glycine. Like prostatic binding protein, proline-rich polypeptide is a characteristic component of the rat ventral prostate and localized primarily in the intraluminal secretion of this gland. In intact adult male rats the cytosol of a whole gland contains 0.70 +/- 0.15 (S.D.) mg of the polypeptide, as measured by radial immunodiffusion or 2.6 +/- 0.5% of (S.D.) of the total protein. This amount decreases gradually after castration and becomes undetectable after 8 days. Androgen treatment, on the other hand, results in a rapid stimulation, while estradiol and progesterone are ineffective. Proline-rich polypeptide is markedly more androgen-dependent than prostatic binding protein, and promises to be an interesting end point for studies on the mechanism of action of androgens.

Amino Acids↗
PubMed1987

Radioautographic tracing of 3H-proline in the endodermal cells of the parietal yolk sac as an indicator of the biogenesis of basement membrane components.

The biogenesis of basement-membrane components was investigated in the endodermal cells of the rat parietal yolk sac in 12.5-day pregnant rats; 3H-proline was injected into conceptuses. After various time intervals, the parietal yolk sac, including endodermal cells and the associated Reichert's membrane, was removed and processed for electron-microscopic radioautography. Silver grains were counted over endodermal cell organelles and Reichert's membrane. At 2 and 5 min after 3H-proline injection, endodermal cells showed heavy labeling in rough endoplasmic reticulum (rER). Silver grain density over the rER decreased from 2 to 20 min and then remained at a plateau. Grain density was moderate over the Golgi apparatus initially but rose to a peak at 2 hr and decreased by 4 hr and later. Grain density was negligible over secretory granules at 2 and 5 min and increased moderately with time to reach a maximum at 8 hr. Thus, radioautographic peaks occurred sequentially in rER, Golgi apparatus, and secretory granules. By 4 hr and later, silver grains accumulated over Reichert's membrane. These results indicated that endodermal cells incorporated labeled proline into substances which were processed from the rER through the Golgi apparatus, transported from there to the cell surface by secretory granules, and released for export to Reichert's membrane. To clarify the nature of the exported substances, the amount of label present in proline and hydroxyproline residues after 3H-proline injection was measured in Reichert's membrane with or without the associated endodermal cells. Within the cells, 61.8% of the labeled proteins were classified as "sedentary" and 38.2% as "exportable." Of the label exported to Reichert's membrane, 66.3% consisted of type IV collagen and the rest of other basement-membrane components. The results obtained with this model suggest that basement-membrane proteins, including type IV collagen, are elaborated by the associated cells through the classical pathway: rER-Golgi apparatus-secretory granules.

Animals↗
PubMed1986

The effect of streptozotocin on the secretory activity of ameloblasts in rat incisor as revealed by radioautography after 3H-proline administration.

The effect of a diabetogenic dose of streptozotocin on the secretory activity of ameloblasts was investigated in the rat incisor by radioautography. One group of male Sprague-Dawley rats was injected intravenously with streptozotocin in citrate buffer (pH 4.5). One hour later, this group was again injected intravenously with 3H-proline (2 mCi/kg). A control group of animals was injected with 3H-proline only. All the animals were sacrificed in groups of three at 5 min, 1 h, 2 h, 4 h and 8 h after 3H-proline injection by perfusion with 3% phosphate-buffered formaldehyde followed by an additional perfusion with 2.5% phosphate-buffered glutaraldehyde. The incisors were extracted with the jaws, demineralized, and prepared for radioautographic observations and analysis. The principal effects of streptozotocin were as follows: There was an inhibition of 3H-proline incorporation into the secretory ameloblasts at 5 min after injection. This was followed by a larger uptake and a slower passage of the label out of the cells into the enamel matrix than that seen in the control sample. Finally, there was a slower secretion of labeled proteins out of Tomes' processes between 1 and 4 h after injection. Therefore, streptozotocin had a temporary inhibitory effect on the incorporation and secretion of 3H-proline by the secretory ameloblasts of the rat incisor. This effect was present for about 4 h and was completely reversed 9 h after streptozotocin injection.

Ameloblasts↗
PubMed1986

Osteodentin formation in rat incisor as visualized by radioautography after 3H-proline administration.

Osteodentin formation was studied in rat incisor pulp after adriamycin administration. Male Sprague Dawley rats (100 +/- 5 gm) were injected intravenously with adriamycin (5 mg/kg body weight), and after 7 days they were again injected intravenously with 3H-proline (3 microCi/gm). These animals were killed in groups of three from 5 minutes to 4 hours after proline injection by perfusion with 3% phosphate-buffered formaldehyde followed by 2.5% phosphate-buffered glutaraldehyde. Control animals injected with only physiological saline, and 7 days later with 3H-proline (3 microCi/gm), and were killed at the same time intervals. Radioautography on sections showing osteodentin formation revealed that at 5 minutes after 3H-proline injection the labeling was located over the cells associated with the osteodentin matrix. At 1 hour after injection the labeling was located over the cells and the matrix, while at 4 hours the labeling was seen only over the matrix. It therefore appears that at least a proline-containing component of the osteodentin matrix is synthesized and secreted by the cells associated with it.

Animals↗
PubMed1990

Synthesis and peptide bond orientation in tetrapeptides containing L-azetidine-2-carboxylic acid and L-proline.

The role of the amino acid proline in influencing the secondary and tertiary structure of proteins and polypeptides has been an area of active study for many years. We have investigated this problem by incorporating the four-membered ring amino acid, azetidine-2-carboxylic acid, into some proline polypeptides. An adjunct to the synthesis of the peptides was the synthesis of azetidine-2-carboxylic acid and its resolution. We developed an improved synthesis of N-benzhydryl-2-carbobenzyloxy azetidine, an essential intermediate required for the synthesis of L-azetidine-2-carboxylic acid. This amino acid was subsequently obtained via the partial hydrogenation of the N-benzhydryl compound, under mild conditions. Our ability to isolate the intermediate N-benzhydryl-2-carboxylic acid demonstrated that the rate of cleavage of the O-benzyl ester group in this molecule is faster than the cleavage of the N-benzhydryl group. The tetrapeptides, Boc-(L-Pro)3-L-Aze-Opcp, and Boc-(L-Aze-L-Pro)2-Opcp (Boc: t-butoxycarbonyl; Pro: proline; Aze: azetidine-2-carboxyl acid; Opcp: pentachlorophenyl), were prepared using traditional solution peptide synthesis. They were characterized by direct chemical ionization-mass spectrometry, CD spectra, and 13C- and 1H-nmr spectroscopy. The assessment of the secondary structure of the two peptides using the methods noted above has led us to conclude that the compound Boc-(L-Aze-L-Pro)2-Opcp, in trifluoroethanol, has an all-cis peptide bond conformation with phi and psi torsion angles compatible with a left-handed helix. The secondary structure assessment of the peptide Boc-(L-Pro)3-L-Aze-Opcp, in chloroform or trifluoroethanol, leads to an assignment of both cis and trans peptide bonds as being present in the peptide. We have interpreted this latter finding as indicating that the introduction of the azetidine group into a peptide containing three consecutive proline residues in a linear sequence perturbs the normal proline peptide secondary structure in this tetrapeptide.

Azetidinecarboxylic Acid↗
PubMed1992

Flexible-geometry conformational energy maps for the amino acid residue preceding a proline.

Previously calculated conformational energy maps suggest that the alpha-helical conformation for the residue preceding a proline is disfavored relative to the extended conformation by more than 7 kcal/mol. In known protein structures this conformation is observed, however, to occur for about 9% of all prolines. In addition, introduction or removal of prolines at theoretically unfavorable positions in proteins and peptides can have modest effects on stability and structure. To investigate the discrepancy between calculation and experiment, we have determined how the conformation of the proline affects the calculated energy. We have also explored the effect of bond length and bond angle relaxation on the conformational energy map. The conformational energy of the preceding residue is found to be unaffected by the conformation of the proline, but the effect of allowing covalent bond relaxation is dramatic. If bond lengths and angles, and dihedral angles within the pyrrolidine ring, are allowed to relax, a calculated energy difference between the alpha and beta conformations of 1.1 kcal/mol is obtained, in reasonable agreement with experiment. The detailed shape of the calculated energy surface is also in excellent agreement with the observed conformational distributions in known protein structures.

Amino Acid Sequence↗
PubMed2005

Synthesis of fluorescent label, DBD-beta-proline, and the resolution efficiency for chiral amines by reversed-phase chromatography.

DBD-d(and l)-beta-proline, new fluorescent chiral derivatization reagents, were synthesized from the reaction of 4-(N,N-dimethylaminosulfonyl)-7- fl uoro-2,1,3-benzoxadiazole (DBD-F) with beta-proline. The racemic mixture synthesized was separated by a chiral stationary phase (CSP) column, Chiralpak AD-H, with n-hexane-EtOH-TFA-diethylamine (70:30:0.1:0.1) as the mobile phase. The dl-forms were decided according to the results obtained from a circular dichroism (CD) detector after separation by the CSP column. The fractionated enantiomers reacted with chiral amine to produce a couple of diastereomers. The labeling proceeded in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine as the activation reagents. The reaction conditions were mild and no racemization occurred during the diastereomer formation. The resulting diastereomers fluoresced at around 570 nm (excitation at around 460 nm). Good linearity of the calibration curves was obtained in the range 1-75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was compared with the diastereomers derived from DBD-d(or l)-proline. The resolution values (Rs) obtained from the diastereomers of three chiral amines with DBD-d(or l)-beta-proline were higher than those derived from DBD-d(or l)-proline, e.g. dl-phenylalanine methylester (dl-PAME), 2.23 vs 1.37; (R)(S)-1-phenylethylamine [(R)(S)-PEA], 2.09 vs 1.13; and (R)(S)-1-(1-naphthyl)ethylamines [(R)(S)-NEA], 5.19 vs 1.23. The results suggest that the position of COOH group on pyrrolidine moiety in the structures is one of the important factors for the efficient separation of a couple of the diastereomers.

Amines↗
PubMed2006

5-(pyrrolidin-2-yl)-1H-tetrazole and 5-[(pyrrolidin-2-yl)methyl]-1H-tetrazole: proline surrogates with increased potential in asymmetric catalysis.

Catalytic enantioselective methodology has dramatically been enriched by the re-discovery of the simple amino acid proline as a chiral catalyst in the year 2000. Although no catalyst offers such a simple and broad access to quite complex reaction products, as does proline, its synthetic potential is not unrestricted, what is especially connected to its poor solubility in organic media. Exchange of the carboxylic moiety by a tetrazole unit leads to proline surrogates, that by far outperform proline with respect to yield, enantioselectivity, reaction time, substrate and solvent scope, catalyst loading, and stoichiometry of the compounds used in excess. These factors are discussed and critically compared with selected representative proline-catalyzed reactions.

Catalysis↗
PubMed1985

Effects of bleomycin on 14C-proline uptake, its incorporation into proteins and hydroxylation in collagenous proteins of hamster lung slices.

The uptake of 14C-proline by lung slices and its incorporation into trichloroacetic-acid (TCA)-insoluble proteins and hydroxylation in collagenous proteins were studied. They were found to be linearly increased with the incubation time for as long as 8 h. The effects of different concentrations of bleomycin on these parameters were subsequently investigated. Bleomycin at 10(-3) M significantly reduced the uptake of 14C-proline to 75% of the control value. The incorporation of 14C-proline into TCA-insoluble proteins was reduced to 84%, 78% and 75% of the control at 2.5 X 10(-4) M, 5 X 10(-4) M and 10(-3) M concentrations of bleomycin, respectively. Bleomycin failed to alter the proline pool size of the lung slices at any of these concentrations. In addition, bleomycin also inhibited the hydroxylation of 14C-proline into collagen to 72% and 69% of the control at 5 X 10(-4) M and 10(-3) M, respectively. These findings show that bleomycin-induced increases in collagen synthesis in vivo is probably secondary to inflammatory reactions of the lung and bleomycin per se has little effect on increased collagen synthesis responsible for lung fibrosis.

Animals↗
PubMed1978

[Differences in proline metabolism of a peptide alkaloid producing and a nonproducing strain of Claviceps purpurea].

Two strains (pepty 695 and pur 221) of Claviceps purpurea have been used to study the dependence of proline turnover on incubation time (1, 3, 6, and 12 hours) under different culture conditions (synthetic saccharose-citrate medium NL 720 and complex wort medium M 107) by means of tracer technique. In the saprophytically ergotoxine producing strain pepty 695 the proline is utilized in the protein and alkaloid biosyntheses. On the other hand, under non-producing conditions (M 10) the radioactivity of proline-14C is incorporated into many fractions and finally into CO2. In submerged culture (NL 720) the Claviceps strain pur 221 which does not produce ergolines uses proline only to a small extent in the protein biosynthesis, while most of this amino acid is not metabolized. The extent of uptake of proline is not correlated with the alkaloid synthesis. For comparison the turnover of L-glutamate-14C and D,L-ornithine-1-14C by strain pepty 695 cultivated in the NL 720 medium has been studied.

Claviceps↗
PubMed1979

Effects of L-proline and some of its analogs on retinal spreading of depression.

L-Proline inhibits glutamate-based spreading depressions (SDs) at low concentrations (2--2.5 mM) and promotes K+-based SDs at higher concentrations (5 mM). The inhibition of glutamate-based SDs was postulated to be due to competition of L-glutamate and L-proline for glutamate receptors on somatic and dendritic plasma membranes. The binding of proline to glutamate receptors was furthermore postulated to result in a release of K+ from the intracellular compartment, enhancing the extracellular K+ concentration sufficiently to promote K+-based SDs. A proline analog, L-baikiain, containing a double bond and one more C atom in the ring structure than proline had similar effects as the latter amino acid, but an analog, L-azetidine-2-carboxylic acid, with one less C atom in the ring had little effect on SD in the retina.

Animals↗
PubMed2007

A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase.

Analysis of the three-dimensional structures of three closely related mesophilic, thermophilic, and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Clostridium beijerinckii (CbADH), Entamoeba histolytica (EhADH1), and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro100) might be crucial for maintaining the thermal stability of EhADH1. To determine whether proline substitution at this position in TbADH and CbADH would affect thermal stability, we used site-directed mutagenesis to replace the complementary residues in both enzymes with proline. The results showed that replacing Gln100 with proline significantly enhanced the thermal stability of the mesophilic ADH: DeltaT(1/2) (60 min) = + 8 degrees C (temperature of 50% inactivation after incubation for 60 min), DeltaT(1/2) (CD) = +11.5 degrees C (temperature at which 50% of the original CD signal at 218 nm is lost upon heating between 30 degrees and 98 degrees C). A His100 --> Pro substitution in the thermophilic TbADH had no effect on its thermostability. An analysis of the three-dimensional structure of the crystallized thermostable mutant Q100P-CbADH suggested that the proline residue at position 100 stabilized the enzyme by reinforcing hydrophobic interactions and by reducing the flexibility of a loop at this strategic region.

Alcohol Dehydrogenase↗
PubMedDate not supplied

The use of proline as a nitrogen source causes hypersensitivity to, and allows more economical use of 5FOA in Saccharomyces cerevisiae.

The use of proline as a nitrogen source causes hypersensitivity to 5-fluoro-orotic acid (5FOA) and allows up to 40-fold less of this drug to be used to select for the loss of URA3 function in Saccharomyces cerevisiae. 5FOA hypersensitivity is presumably due to the absence of nitrogen catabolite repression when proline is substituted for (NH4)2SO4 as a nitrogen source. There are two constraints to the use of the proline-5FOA combination: (1) S288c genetic background strains are hypersensitive to 5FOA when grown in proline as a nitrogen source but at least one other genetic background is resistant to low levels of 5FOA under these conditions. (2) The addition of some nutritional supplements confers phenotypic resistance to the 5FOA-proline combination.

Culture Media↗
PubMed1993

Suppression in mitochondrial electron transport is the prime cause behind stress induced proline accumulation.

Exposure of six day old rice (Oryza sativa) seedlings to salt or cadmium stress lead to an increase in the level of proline with a simultaneous decline in the mitochondrial electron transport activity. Mitochondrial electron transport inhibitors - rotenone, antimycin A or potassium cyanide also stimulated proline accumulation in rice seedlings with a concurrent decline in the mitochondrial electron transport activity. Four to five fold enhancement in proline level was noted in seedlings after 48 h exposure to electron transport inhibitors. A significant rise in the level of NADH was also noted in seedlings exposed to salt stress, cadmium stress or any of the electron transport inhibitors. These results show for the first time that the suppression in the mitochondrial electron transport activity lead to proline accumulation. Our results also suggest that the increase in the ratio of NADH to NAD+ due to the suppression in mitochondrial electron transport might be the prime reason behind proline accumulation in plants exposed to environmental stresses.

Antimycin A↗
PubMed1996

Effect of serine O-glycosylation on cis-trans proline isomerization.

In order to examine the ability of an O-glycosylated serine residue preceding proline to stabilize a cis amide conformation in a fashion similar to that observed with aromatic amino acid residues, we prepared a series of glycosylated analogs of a small linear peptide which we have previous reported to contain a cis conformation of an amide bond between tyrosine and proline. The glycopeptides were prepared by incorporating glycosylated N alpha- (fluoren-9-yl) methoxycarbonyl (Fmoc) amino acids into a standard solid phase peptide synthesis protocol. The peptides and glycopeptides were analyzed using 2-dimensional NMR spectroscopy. Unlike the case where the residue preceding proline was tyrosine, no signals corresponding to a cis proline conformation were detected in the spectra of the two glycopeptides containing serine O-glycosylated with either beta-linked N-acetyl glucosamine or alpha-linked N-acetyl galactosamine in the position preceding proline.

Acetylglucosamine↗
PubMed2001

Proline suppresses Rubisco activity by dissociating small subunits from holoenzyme.

Proline caused irreversible inhibition (involving reduction in V(max) without altering K(m) for RuBP) in Rubisco activity. Proline-induced suppression in Rubisco activity did not exceed beyond approximately 65% of the original activity even upon exposure to higher levels of proline for prolonged duration. However, NaCl-induced reduction in Rubisco activity was reversible. Native PAGE analysis of Rubisco-incubated with proline showed the presence of two distinct bands corresponding to approximately 430 and approximately 28 kDa, but that incubated with NaCl showed a single band. SDS-PAGE analysis revealed that the approximately 430- and approximately 28-kDa bands represent octamers of large subunits and dimers of small subunits, respectively. These results demonstrated for the first time that proline suppresses Rubisco activity by bringing about dissociation of the small subunits from the octamer core of large subunits, probably by weakening hydrophobic interactions between them.

Catalytic Domain↗
PubMed1997

The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 A resolution: structural characterization of proline-substitution sites for protein thermostabilization.

The crystal structure of oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase, EC 3.2.1.10) from Bacillus cereus ATCC7064 has been refined to 2.0 A resolution with an R-factor of 19.6% for 43,328 reflections. The final model contains 4646 protein atoms and 221 ordered water molecules with respective root-mean-square deviations of 0.015 A for bond lengths and of 3.166 degrees for bond angles from the ideal values. The structure consists of three domains: the N-terminal domain (residues 1 to 104 and 175 to 480), the subdomain (residues 105 to 174) and the C-terminal domain (residues 481 to 558). The N-terminal domain takes a (beta/alpha)8-barrel structure with additions of an alpha-helix (N alpha6') between the sixth strand Nbeta6 and the sixth helix N alpha6, an alpha-helix (N alpha7') between the seventh strand Nbeta7 and the seventh helix N alpha7 and three alpha-helices (N alpha8', N alpha8" and N alpha8'" between the eighth strand Nbeta8 and the eighth helix N alpha8. The subdomain is composed of an alpha-helix, a three-stranded antiparallel beta-sheet, and long intervening loops. The C-terminal domain has a beta-barrel structure of eight antiparallel beta-strands folded in double Greek key motifs, which is distorted in the sixth strand Cbeta6. Three catalytic residues, Asp199, Glu255 and Asp329, are located at the bottom of a deep cleft formed by the subdomain and a cluster of the two additional alpha-helices N alpha8' and N alpha8" in the (beta/alpha)8-barrel. The refined structure reveals the locations of 21 proline-substitution sites that are expected to be critical to protein thermostabilization from a sequence comparison among three Bacillus oligo-1,6-glucosidases with different thermostability. These sites lie in loops, beta-turns and alpha-helices, in order of frequency, except for Cys515 in the fourth beta-strand Cbeta4 of the C-terminal domain. The residues in beta-turns (Lys121, Glu208, Pro257, Glu290, Pro443, Lys457 and Glu487) are all found at their second positions, and those in alpha-helices (Asn109, Glu175, Thr261 and Ile403) are present at their N1 positions of the first helical turns. Those residues in both secondary structures adopt phi and phi values favorable for proline substitution. Residues preceding the 21 sites are mostly conserved upon proline occurrence at these 21 sites in more thermostable Bacillus oligo-1,6-glucosidases. These structural features with respect to the 21 sites indicate that the sites in beta-turns and alpha-helices have more essential prerequisites for proline substitution to thermostabilize the protein than those in loops. This well supports the previous finding that the replacement at the appropriate positions in beta-turns or alpha-helices is the most effective for protein thermostabilization by proline substitution.

Amino Acid Sequence↗